PKC Delta Inhibitors for use as Therapeutics

ABSTRACT

The invention is directed to compounds that are specific inhibitors of PKC delta, and methods and compositions for the treatment and prevention of cancers and other disorders. Compositions comprising compounds of the invention are used to treat cancers such as, for example, carcinoid and neuroendocrine tumors, malignant melanomas, pancreatic, gastrointestinal and lung cancers. Neuroendocrine tumor cell lines of pulmonary and gastrointestinal origin are surprisingly sensitive to PKC delta inhibition by the compounds of the invention. The invention is further directed to methods, compositions and kits containing compounds of the formulas (Ia), (IIa), (IIIa), (IVa), and (V) as disclosed and described in FIGS.  11  and  12.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/428,232 filed Mar. 13, 2015, which is a National Stage submissionunder 35 U.S.C. §371 of International Application No. PCT/US2013/60683filed Sep. 19, 2013, which claims priority to U.S. ProvisionalApplication No. 61/703,081 filed Sep. 19, 2012, the entirety of each ofwhich is specifically incorporated by reference.

RIGHTS IN THE INVENTION

The invention was made with support from the U.S. Government under grantNos. CA112102 and CA141908, awarded by the National Institutes of Health(NIH), and accordingly, the U.S. Government has certain rights in theinvention.

BACKGROUND

1. Field of the Invention

The invention is directed to compounds that are specific inhibitors ofPKC delta, and, in particular, to methods and compositions astherapeutic treatments, and as diagnostics to treat or prevent disorderssuch as cancers.

2. Background of the Invention

Targeting cancer therapeutics towards specific mutations orabnormalities in tumor cells not found in normal tissues has thepotential advantages of high selectivity for the tumor andcorrespondingly low secondary toxicities. At least 30% of all humanmalignancies display activating mutations in the RAS genes, and perhapsanother 60% display other activating mutations in, or over-activity of,p21Ras signaling pathways. It was previously reported that aberrantactivation of Ras produces an absolute dependency upon PKCdelta-mediated survival pathways (Xia S, Forman L W, & Faller D V 2007Protein Kinase C{delta} is required for survival of cells expressingactivated p21RAS. J Biol. Chem. 282 13199-13210; Xia S, Chen Z, Forman LW, & Faller D V 2009 PKC delta survival signaling in cells containing anactivated p21Ras protein requires PDKI. Cell Signal. 21 502-508). Overactivity of p21Ras signaling therefore sensitizes tumor cells toapoptosis induced by suppression of PKC delta, whereas suppression ofPKC delta is not toxic to cells with normal levels of p21Ras activity orsignaling (Chen C Y & Faller D V 1995 Direction of p21Ras-generatedsignals towards cell growth or apoptosis is determined by protein kinaseC and Bcl-2. Oncogene 111487-1498; Xia S, Forman L W, & Faller D V 2007Protein Kinase C{delta} is required for survival of cells expressingactivated p21RAS. J Biol. Chem. 282 13199-13210; Chen C Y & Faller D V1996 Phosphorylation of Bcl-2 protein and association with p21 (Ras) inRas-induced apoptosis. J. Biol. Chem. 271 2376-2379; Chen C Y, Liou J,Forman L W, & Faller D V 1998a Correlation of genetic instability andapoptosis in the presence of oncogenic Ki-Ras. Cell DeathDifferentiation. 5 984-995; Chen C Y, Liou J, Forman L W, & Faller D V1998b Differential regulation of discrete apoptotic pathways by Ras. J.Biol. Chem. 273 16700-16709; Chen C Y, Juo P, Liou J, Yu Q, Blenis J, &Faller D V 2001 Activation of FADD and Caspase 8 in Ras-mediatedapoptosis. Cell Growth Differ. 12 297-306; Liou J S, Chen C Y, Chen J S,& Faller D V 2000 Oncogenic Ras mediates apoptosis in response toprotein kinase C inhibition through the generation of reactive oxygenspecies. J. Biol. Chem. 275 39001-39011; Liou J S, Chen J-C, & Faller DV 2004 Characterization of p21Ras-mediated apoptosis induced by ProteinKinase C inhibition and application to human tumor cell lines. J. CellPhysiol. 198 277-294). This tumor-susceptibility designated“Ras-mediated apoptosis” can be exploited for specific targeted cancertherapeutics.

Bronchopulmonary, gastrointestinal and pancreatic neuroendocrine tumorsare rare tumors originating from neuroendocrine tissues (Oberg K 1999Neuroendocrine gastrointestinal tumors—a condensed overview of diagnosisand treatment. Ann. Oneal. 10 Suppl 2:S3-8. S3-S8). Clinical symptomsare often caused by the production of hormonally active substances bythe tumor such as serotonin, gastrin, insulin, vasoactive intestinalpeptide, pancreatic polypeptide, or substanceP. Chromogranin A isproduced by 80-100% of neuroendocrine tumors and serves as a reliablebiochemical marker. The disease can be cured by early surgery, but thevast majority of tumors have metastases at the time of diagnosis, whichmakes palliation the cornerstone of management. Debulking surgery, liverartery embolization, and chemotherapy aim at tumor mass reduction,whereas somatostatin analogues and IFN are used for control of symptoms(Arnold R, Simon B, & Wied M 2000 Treatment of neuroendocrine GEPtumours with somatostatin analogues: a review. Digestion. 62 Suppll84-91; Frank M, Klose K J, Wied M, Ishaque N, Schade-Brittinger C, &Arnold R 1999 Combination therapy with octreotide and alpha-interferon:effect on tumor growth in metastatic endocrine gastroentero pancreatictumors. Am. J. Gastroenterol. 94 1381-1387).

Radioactively-labeled somatostatin analogues have been used in trials,with response rates 30% (Arnold R, Wied M, & Behr T H 2002 Somatostatinanalogues in the treatment of endocrine tumors of the gastrointestinaltract. Expert. Opin. Pharmacother. 3 643-656).

Response rates of cytoreductive approaches to such cancers are generallybelow 60%, however, and their use has limited utility because long-termresponses are not maintained (Oberg K 2001 Chemotherapy and biotherapyin the treatment of neuroendocrine tumours. Ann. Oncol. 12 Suppl2:S111-4.). Accordingly, new and more effective approaches are thereforeneeded in the treatment of neuroendocrine malignancies.

Carcinoid and other neuroendocrine tumors of the gastrointestinal tractshare a number of the same genetic abnormalities (deletions andmutations) as adenocarcinomas (Leotlela P D, Jauch A, Holtgreve-Grez H,& Thakker R V 2003 Genetics of neuroendocrine 5 and carcinoid tumours.Endocr. Relat Cancer. 10 437-450; Leotlela et al. 2003; Arber N, NeugutA I, Weinstein I B, & Holt P 1997 Molecular genetics of small bowelcancer. Cancer Epidemiol. Biomarkers Prev. 6 745-748). Theseabnormalities include activation of Ras directly by mutations,indirectly by loss of Ras-regulatory proteins such as NF-1, or viaconstitutive activation of downstream effector pathways of Ras, such asPI3K and Raf/MAP kinase. For example, activation of H-Ras and Ki-Ras aredetected in a significant fraction of carcinoid and othergastrointestinal tumors (65% and 10%, respectively) (Liedke M, KarnbachC, Kalinin V, Herbst B, Frilling A, & Broelsch C E 1998 [Detection ofH-ras and K-ras in tumors of gastrointestinal-pancreatic system].Langenbecks Arch. Chir Suppl Kongressbd. 115 255-259; Maitra A, KruegerJ E, Tascilar M, Offerhaus G J, Angeles-Angeles A, Klimstra D S, HrubanR H, & Albores-Saavedra J 2000 Carcinoid tumors of the extrahepatic bileducts: a study of seven cases. Am. J. Surg. Pathol. 24 1501-1510). Rascan also be activated in carcinoid and other neuroendocrine by eitherpoint mutation or loss of regulators of Ras, such as RassF1A or NF-1(Liu L, Broaddus R R, Yao J C, Xie S, White J A, Wu T T, Hamilton S R, &Rashid A 2005 Epigenetic alterations in neuroendocrine tumors:methylation of RAS association domain family 1, isoform A and p16 genesare associated with metastasis. Mod. Pathol. 18 1632-1640; Stancu M, WuT T, Wallace C, Houlihan P S, Hamilton S R, & Rashid A 2003; Geneticalterations in goblet cell carcinoids of the vermiform appendix andcomparison with gastrointestinal carcinoid tumors. Mod. Pathol. 161189-1198; Bausch B, Borozdin W, Mautner V F, Hoffmann M M, Boehm D,Robledo M, Cascon A, Harenberg T, Schiavi F, Pawlu C, et al. 2007Germline NF1 mutational spectra and loss-of-heterozygosity analyses inpatients with pheochromocytoma and neurofibromatosis type 1. J. Clin.Endocrinol. Metab. 92 2784-2792). The Raf/mitogen-activated proteinkinase (Raf/MAP kinase), or the MAP kinases directly downstream of Raf,are frequently activated in carcinoid tumors (Tannapfel A, Vomschloss S,Karhoff D, Markwarth A, Hengge U R, Wittekind C, Arnold R, & Horsch D2005 BRAF gene mutations are rare events in gastroenteropancreaticneuroendocrine tumors. Am. J. Clin. Pathol. 123 256-260; Karhoff D,SauerS, Schrader J, Arnold R, Fendrich V, Bartsch D K, & Horsch D 2007Rap1/B-Raf signaling is activated in neuroendocrine tumors of thedigestive tract and Raf kinase inhibition constitutes a putativetherapeutic target. Neuroendocrinology 85 45-53; Perren A, Schmid S,Locher T, Saremaslani P, Bonvin C, Heitz P U, & Komminoth P 2004 BRAFand endocrine tumors: mutations are frequent in papillary thyroidcarcinomas, rare in endocrine tumors of the gastrointestinal tract andnot detected in other endocrine tumors. Endocr. Relat Cancer 11 855-860;Kunnimalaiyaan M & Chen H 2006 The Raf-1 pathway: a molecular target fortreatment of select neuroendocrine tumors? Anticancer Drugs 17 139-142).The PI3K pathway is activated in carcinoid tumors from deletion of thetumor suppressor gene PTEN (phosphatase and tensin homologue). Loss ofPTEN in neuroendocrine and carcinoid tumors, increases in frequency withthe loss of differentiation in the tumor (Wang G G, Yao J C, Worah S,White J A, Luna R, Wu T T, Hamilton S R, & Rashid A 2005 Comparison ofgenetic alterations in neuroendocrine tumors: frequent loss ofchromosome 18 in ileal carcinoid tumors. Mod. Pathol. 18 1079-1087), andloss of PTEN expression may represent an important step in theprogression of neuroendocrine tumors (Wang L, Ignat A, & Axiotis C A2002 Differential expression of the PTEN tumor suppressor protein infetal and adult neuroendocrine tissues and tumors: progressive loss ofPTEN expression in poorly differentiated neuroendocrine neoplasms. Appl.Immunohistochem. Mol. Morphol. 10 139-146).

Gastrointestinal and pulmonary carcinoid tumors are uncommon, butunfortunately are generally refractory to conventional cytotoxicchemotherapeutic and radiotherapeutic approaches. Many targetedtherapeutic approach such as induction of Ras-mediated apoptosis by PKCdelta inhibition, which selectively takes advantage of the veryoncogenic mutations which contribute to the malignancy of the tumor, mayhave potential as a novel and selective therapeutic modality for thesemalignancies.

SUMMARY OF THE INVENTION

The present invention generally relates to compounds which werediscovered to be specific inhibitors of PKC delta, and methods andcompositions thereof. In some embodiments, compositions comprising thesecompounds can be used in a method to treat cancers, e.g., carcinoid andneuroendocrine tumors, malignant melanomas, pancreatic, gastrointestinaland lung cancers. Herein, the inventors have demonstrated that humanneuroendocrine tumor cell lines of pulmonary and gastrointestinal originare surprisingly sensitive to PKC delta inhibition using the compoundsas disclosed herein (Chen, Z., Forman, L. W., Miller, K. A., English,B., Takashima, A., Bohacek, R. A., Williams, R. M., and Faller, D. V.:The proliferation and survival of human neuroendocrine tumors isdependent upon protein kinase C-delta. 2011, Endocrine-Related Cancers,18:759-71).

In certain embodiments, a PKC delta inhibitory compound for use in themethods, compositions and kits as disclosed herein is of the formula(Ia), (IIa), (IIIa), (IVa), or (V) (see FIG. 12). The compounds of theinvention specifically inhibit PKC delta. The invention also providespharmaceutical compositions comprising a therapeutically effectiveamount of a compound of the invention and a pharmaceutically acceptableexcipient. Herein the inventors have demonstrated that a compound asdisclosed herein is a specific inhibitor of PKC delta and produces adose-dependent and time-dependent decrease in cell numbers for a numberof neuroendocrine tumor cell lines: BON1 (foregut carcinoid tumor cellline), CNDT 2.5 and H727 cell lines. Additionally, the inventors havedemonstrated that the compounds as disclosed herein significantlysuppress cell growth and clonogenic capacity of these cell lines.

Accordingly, one aspect of the present invention relates to use of acompound of formula (Ia), (IIa), (IIIa), (IVa), or (V) and itsderivatives and analogues in a method to treat cancers and/or inhibitcell proliferation, for example, bronchopulmonary, gastrointestinal andpancreatic neuroendocrine tumors, malignant melanomas, pancreatic,gastrointestinal and lung cancers. Other aspects of the proposedinvention relate to methods to treat human neuroendocrine tumors. Inparticular, the inventors have demonstrated that inhibition ordownregulation of PKC delta by siRNA, and small molecule inhibitors suchas a compound of formula (Ia), (IIa), (IIIa), (IVa), or (V) canefficiently and selectively repress the growth of human neuroendocrinecells, derived from bronchopulmonary, foregut and hindgut tumorscarcinoid and neuroendocrine tumors, malignant melanomas, pancreatic,gastrointestinal and lung cancers.

Accordingly, in some embodiments a pharmaceutical composition comprisingat least one compound as disclosed herein can be used in a method totreat a cancer in a subject. In some embodiments, a compound asdisclosed herein can be used in a method to treat a cancer in a subjectfor example, a bronchopulmonary cancer, a gastrointestinal cancer or apancreatic neuroendocrine cancer, carcinoid and neuroendocrine tumors,malignant melanomas, pancreatic, gastrointestinal and lung cancers. Insome embodiments, a compound as disclosed herein can be used in a methodto treat a carcinoid and/or neuroendocrine cancer. In some embodiments,a neuroendocrine cancer can be derived from a bronchopulmonary, orforegut or hindgut tumor.

In another embodiment, the present invention can be used as a method oftreating a pathological condition in a subject that is responsive toinhibition of PKC delta, comprising administering to the subject atherapeutically or prophylactically effective amount of a compound, or apharmaceutical composition thereof. In certain embodiments, the compoundor pharmaceutical composition is administered orally. In otherembodiments, the compound or pharmaceutical composition is administeredparenterally (e.g., intravenously).

The compounds of the invention can also be used in a method ofinhibiting PKC delta in a subject and a method of treating a subjectwith cancer, for example, a bronchopulmonary cancer, a gastrointestinalcancer or a pancreatic neuroendocrine cancer, malignant melanomas,pancreatic, gastrointestinal and lung cancers in which a therapeuticallyeffective amount of an inventive compound, or a pharmaceuticalcomposition thereof, is administered to the subject.

The invention further relates to the use of the compounds of theinvention for the manufacture of a medicament for treating pathologicalconditions responsive to inhibition of PKC delta for treating a subjectwith cancer, for example, a bronchopulmonary cancer, a gastrointestinalcancer or a pancreatic neuroendocrine cancer, malignant melanomas,pancreatic, gastrointestinal and lung cancers.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE DRAWINGS

This patent or application contains at least one drawing executed incolor. Copies of this patent or patent application with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee.

FIGS. 1A-1C. Effects of PKC delta knockdown by SiRNA on proliferation ofhuman neuroendocrine tumor BON1 and CNDT cells. BON1 (FIG. 1A) and CNDT2.5 (FIG. 1B) cells were grown to 50% confluence in 96-well plates andthen treated with PKC delta-siRNA or scrambled siRNA (sc-siRNA). Thecorresponding solvent equivalent volumes were used as vehicle controls(Control). After 48, 72, and 96 hours of treatment, cell number wasevaluated by MTS assay. (FIG. 1C) Immunoblot analyses showdown-regulation of PKC delta 72 hr after treatment withlentivirus-transfected PKC delta-targeting siRNA versus scrambledsiRNA-treated controls. Lentiviral PKC delta-targeting siRNA inhibitedPKC delta protein expression, as determined by immunoblotting.

FIGS. 2A-2D. Effects of PKC delta knockdown by PKC delta-specific shRNAlentivirus on proliferation of human neuroendocrine tumor cells. BON1(FIG. 2A), CNDT 2.5 (FIG. 2B) and H727 (FIG. 2C) cells were grown to 50%confluence in 96-well plates and then infected with PKCdelta-shRNA-Lentivirus or scrambled shRNA Lentivirus (vector). Cellexposed to mock lentiviral infection (vehicle) also served as controls.After 24, 48, and 15 72 hours of treatment, cell proliferation wasevaluated by MTS assay. Control values were normalized to 100%. Errorbars represent SEM. P values for comparison between control (scrambledshRNA) lentivirus and PKC delta-shRNA lentivirus effects on cell numberreached significance at 24 hr of exposure (p<0.001) for all cell lines,and remained significant at the 48 and 72 hr time points. (FIG. 2D)Immunoblot analyses of PKC delta protein 72 hr after exposure tolentivirus-transfected PKC delta-targeting shRNA compared to infectionwith lentivirus containing a scrambled shRNA-treated (Sc-shRNA), ormock-infected controls.

FIGS. 3A-3C. Cytotoxic effects of PKC delta knockdown byshRNA-lentivirus on human neuroendocrine tumor cell lines. BON1 (FIG.3A), CNDT 2.5 (FIG. 3B) and H727 (FIG. 3C) cells were grown to 50%confluence in 96-well plates and then infected with PKCdelta-shRNA-lentivirus or scrambled siRNA lentivirus (vector). Cellexposed to mock lentiviral infection (vehicle) also served as controls.After 24, 48 and 72 hours of treatment, cell cytotoxicity was evaluatedby LDH-release assay. Total maximal LDH release is assigned thearbitrary value of 100%. Error bars represent SEM. P values forcomparison between control (scrambled shRNA) lentivirus and PKCdelta-shRNA lentivirus effects on LDH release reached significance at 24hr of exposure (p<0.004) for all cell lines, and remained significant atthe 48 and 72 hr time points.

FIG. 4. Effects of PKC delta inhibitors on proliferation of humanneuroendocrine tumor cell lines. Cells were grown to 80% confluence in96-well plates and then treated with vehicle control (DMSO), Rottlerinor KAM1 at 5, 10, 20 or 40 μM. The corresponding equivalent volumes ofsolvent were used as vehicle controls. After 72 hours of treatment, cellgrowth was evaluated by MTT assay. Control values were normalized to100%. P values for comparison between control (vehicle) and Rottlerineffects on cell number reached significance at 24 hr of exposure(p<0.004) for all cell lines, and remained significant at the 48 and 72hr time points. P values for comparison between control (vehicle) andKAM1 effects on cell number reached significance by 72 hr of exposure(p<0.02) for all cell lines.

FIG. 5 illustrates the structure of KAM1, a rottlerin/staurosporinechimera.

FIGS. 6A-6B. Cytotoxic effects of PKC5 inhibitors on humanneuroendocrine tumor cell lines. H727 cells were grown to 50% confluencein 96-well plates and then exposed to Rottlerin (FIG. 6A) or KAM1 (FIG.6B) at the concentrations indicated. Cells exposed to vehicle aloneserved as controls. After 24, 48 and 72 hours of treatment, cellcytotoxicity was evaluated by LDH-release assay. Baseline LDH release(vehicle treatment) values were subtracted at each time point. Totalmaximal LDH release is assigned the arbitrary value of 100%. Error barsrepresent SEM. P values for comparison between control (vehicle) andRottlerin or KAM1 effects on LDH release reached significance by 24 hrof exposure (p<0.003), and remained significant at the 48 and 72 hr timepoints.

FIGS. 7A-7D. Longer term exposure of human neuroendocrine tumor celllines to PKC delta-inhibitors. BON1 cells (FIGS. 7A and 7C) or CNDTcells (FIGS. 7B and 7D) were exposed to rottlerin at a sub-optimalconcentration (10 μM). Cells exposed to vehicle alone served ascontrols. At the indicated time points, cell numbers were estimated byMTS assays. In cultures depicted in panels A and B, media was notchanged. In cultures depicted in panels C and D, fresh media containingthe PKC delta inhibitor was replaced after 72 hr of exposure (arrows).Error bars represent SEM. (Fall off in cell numbers in control culturesat the longest time points likely reflect overgrowth observed in thecontrol cultures.)

FIG. 8. Duration of exposure to PKC delta inhibitors needed to inhibittumor cell proliferation. BON1 cells were grown to 30% confluence andthen treated with vehicle as control (vehicle), or rottlerin at 10 for6, 12, 24, 48 or 72 hr. Media without inhibitor was replaced and cellnumbers were estimated by MTS assay at 24, 48 and 72 hr. Shown here arethe results at 72 hr of culture after each washout interval. Error barsrepresent SEM. Differences in proliferation between rottlerin- andvehicle-treated cultures became statistically significant by 24 hr ofexposure, and remained significant for all longer periods of exposure.

FIG. 9. Effects of PKC delta inhibitor on tumor cell clonogeniccapacity. H727 cells were grown to 30% confluence and then treated withvehicle as control (vehicle), or rottlerin at 10 for 6, 12, 24, 48 or 72hr. Viable cells were enumerated and re-plated in media withoutinhibitor, and colony numbers were quantitated 96 hr later. Error barsrepresent SEM. P value for comparison of DMSO control and rottlerineffects on clonogenic capacity reached significance (p=0.0051) at 6 hrof exposure and remained significant for all subsequent exposure times.

FIGS. 10A-10B. Ras signaling in neuroendocrine tumor cell lines. FIG.10A illustrates p21Ras activity in neuroendocrine tumor cell lines.Nuclear-free lysates containing a total of 400 μg of protein from eachindicated cell type were used for analysis of Ras activity by Raf-RBDpull-down of GTP-bound p21Ras. Equal loading was demonstrated byre-probing the blot with anti-actin antibody. Pan-p21Ras proteinexpression levels were also analyzed. Lanes 1-5 represent lysates from:NIH/3T3 (negative control), NIH/3T3-Ras (positive control), BON1, H727,and CNDT cells, respectively. FIG. 10B illustrates the activation of Rassignaling pathways in neuroendocrine tumor cell lines. Cell lysates fromnegative control MCF cells (lane 1); positive control MCF-10-Ras cells(lane 2); BON1 cells (lane 3); H727 cells (lane 4); and CNDT cells (lane5) were separated by SDS polyacrylamide gel electrophoresis, transferredto a membrane, and immunoblotted with antibodies against ERK,phospho-ERK, AKT, phospho-Thr308 AKT, and GAPDH (as a loading control).

FIGS. 11A-11M show preferential embodiments of compounds for the methodsand composition of the invention.

FIGS. 12A-12D and 12F show preferential embodiments of compounds for themethods and composition of the invention. FIG. 12E shows a syntheticscheme for one embodiment of the invention.

DESCRIPTION OF THE INVENTION

As discussed above, there remains a need for treatment of malignancies.The present invention provides compounds of general formulae (Ia),(IIa), (IIIa), (IVa), and (V) which are useful as inhibitors of PKCdelta, and thus are useful for the treatment of diseases or disordersassociated with increased activity of PKC delta, and/or increased oroverexpression of PKC delta. In certain embodiments, the inventivecompounds are useful in the treatment of cancer in a subject, forexample, a bronchopulmonary cancer, a gastrointestinal cancer or apancreatic neuroendocrine cancer.

In some embodiments, the cancer is a carcinoid and/or neuroendocrinecancer, malignant melanoma, pancreatic, gastrointestinal or lung cancer.In some embodiments, a neuroendocrine cancer is of pulmonary andgastrointestinal origin, for example, a cancer is derived from abronchopulmonary, or foregut or hindgut tumor.

DEFINITIONS

Before further description of the present invention, and in order thatthe invention may be more readily understood, certain terms are firstdefined and collected here for convenience.

Certain compounds of the present invention, and definitions of specificfunctional groups are described in more detail below. For purposes ofthis invention, the chemical elements are identified in accordance withthe Periodic Table of the Elements, CAS version, Handbook of Chemistryand Physics, 75th Ed., inside cover, and specific functional groups aregenerally defined as described therein. Additionally, general principlesof organic chemistry, as well as specific functional moieties andreactivity, are described in Organic Chemistry, Thomas Sorrell,University Science Books, Sausalito: 1999, the entire contents of whichare incorporated herein by reference. Furthermore, it will beappreciated by one of ordinary skill in the art that the syntheticmethods, as described herein, utilize a variety of protecting groups. Bythe term “protecting group,” has used herein, it is meant that aparticular functional moiety, e.g., C, 0, S, or N, is temporarilyblocked so that a reaction can be carried out selectively at anotherreactive site in a multifunctional compound. In certain embodiments, aprotecting group reacts selectively in good yield to give a protectedsubstrate that is stable to the projected reactions; the protectinggroup must be selectively removed in good yield by readily available,preferably nontoxic reagents that do not attack the other functionalgroups; the protecting group forms an easily separable derivative (morepreferably without the generation of new stereogenic centers); and theprotecting group has a minimum of additional functionality to avoidfurther sites of reaction. As detailed herein, oxygen, sulfur, nitrogen,and carbon protecting groups may be utilized. Exemplary protectinggroups are detailed herein, however, it will be appreciated that thepresent invention is not intended to be limited to these protectinggroups; rather, a variety of additional equivalent protecting groups canbe readily identified using the above criteria and utilized in themethod of the present invention. Additionally, a variety of protectinggroups are described in Protective Groups in Organic Synthesis, ThirdEd. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York:1999, the entire contents of which are hereby incorporated by reference.Furthermore, a variety of carbon protecting groups are described inMyers, A.; Kung, D. W.; Zhong, B.; Movassaghi, M.; Kwon, S. J. Am. Chem.Soc. 1999, 121, 8401-8402, the entire contents of which are herebyincorporated by reference.

The term “PKC delta” is used interchangeably herein with PCK delta orPKC, and refers to the amino acid sequences of substantially purifiedPKC delta obtained from any species, particularly mammalian, includingbovine, ovine, porcine, murine, equine, and preferably human, from anysource whether natural, synthetic, semi-synthetic, or recombinant. PKCdelta (also known in the art as aliases: PRKCD; MAY1; MGC49908; nPKCdelta) is a member of the PKC family.

The term “inhibitor” or “antagonist”, as used herein in reference to aPKC delta antagonist or inhibitor, refers to a molecule which, whenbound to PKC delta, decreases the amount or the duration of the effectof the biological or immunological activity of PKC delta, regardless ofwhether the inhibitor functions indirectly or directly on PKC delta.

The term “PKC delta inhibitor” or “PKC delta antagonist” as used hereinrefers to an agent that reduces or attenuates the biological activity ofthe PKC delta polypeptide in a cell, either by decreasing the activityof the PKC delta polypeptide or by effectively reducing the amount ofPKC delta polypeptide in a cell or by decreasing the enzymatic activityof the PKC delta polypeptide. A “PKC delta inhibitor” thus refers to amolecule having the ability to inhibit a biological function of a nativePKC delta, as well as a mutant PKC delta protein. Compounds that areinhibitors of PKC delta include all solvates, hydrates, pharmaceuticallyacceptable salts, tautomers, stereoisomers, and prodrugs of thecompounds. While preferred PKC delta inhibitors herein specificallyinteract with, e.g. bind to, a PKC delta, molecules that inhibit PKCdelta biological activity by interacting with other members of the PKCdelta signal transduction pathway are also specifically included withinthis definition. Useful PKC delta inhibitors may selectively inhibit PKCdelta, may selectively inhibit calcium-independent or novel PKCisoforms. A preferred PKC delta biological activity inhibited by a PKCdelta inhibitor as disclosed herein is associated with the development,growth, or spread of a tumor or associated with the development orproliferation. Some PKC delta inhibitors may function by more than onemechanism to inhibit overall PKC delta activity in a cell.

The term a “selective” PKC delta inhibitor as used herein refers to anagent that inhibits PKC delta activity with a Ki at least 10-fold less,preferably, at least 100-fold less, than the Ki for inhibition of one ormore other PKC isoforms (e.g., PKC alpha, PKC betta and PKC gamma or anyother).

A “PKC delta targeting treatment” is the use of one or more PKC deltainhibitors to therapeutically reduce PKC delta activity in a cell. A PKCdelta inhibitor may preferably be agents that selectively inhibit PKCdelta. As used herein, an agent that “selectively inhibits” PKC deltameans an agent that reduces the activity of PKC delta more than itreduces the activity of one or more other PKC isoforms.

The term “decreased PKC delta activity” means a substantial decrease bya statistically significant amount in the total PKC delta polypeptideactivity of the PKC delta enzyme as a result of inhibition with a PKCdelta inhibitor compound as disclosed herein as compared to in theabsence of such inhibitor.

The term “biologically active”, as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule, and displays the activity of the molecule in acellular and/or in vivo assay.

The terms “Ras” and “p21Ras” are used interchangeably herein to refer tothe protein product of a Ras gene. The mammalian ras gene familyconsists of the Harvey and Kirsten ras genes (c-Hras1 and c-Kras2), aninactive pseudo gene of each (c-Hras2 and c-K-ras1) and the N-ras gene.The p21Ras protein products of the three ras genes (p21HRas, p21KRas andp21NRas, respectively) differ significantly only in their C-terminal 40amino acids, and each are activated by the same or correspondingactivating mutations. The three ras gene sequences, as well as theirprotein products, for a variety of animals, (e.g. mammals, includinghumans) are well known in the field. Examples of each of the human rasgene coding sequences are provided in International ApplicationW0/20071106424 (see FIG. 8: H-ras in FIG. 8 A, K-ras in FIG. 8B, andN-ras in FIG. 8C), which is incorporated herein in its entirety byreference.

As used herein, the term “activated Ras mutation” refers to the presenceof a genomic mutation in a ras gene which leads to the expression of anactivated form of the Ras protein. The term “wild-type” is used hereinto refer to nucleic acids encoding Ras proteins that do not containactivating mutations, and also to refer to Ras proteins which do notresult from activating mutations.

The term “aberrantly increased Ras signaling” as used herein refers to astatistically significant increase in Ras signaling in one or more cells(e.g. tumor or pre-tumor cells) as measured by a determination of thepercentage of Ras in the activated state and/or activity or one or moredownstream effectors of Ras. Such determination is further made bycomparison of similar measurements made in a similar cells type underappropriate conditions. Increased activity may be surmised by detectionof a known activated Ras mutant, at the nucleic acid or the proteinlevel. Increased activity of Ras may also be surmised by a detectedabnormal increase in the activity and/or presence of a known activatorof Ras or abnormal decrease in known deactivator of Ras, as describedherein. Verification of actual increase in Ras signaling may be used toconfirm such surmisal.

As used herein, the term “subject” or “patient” refers to any mammal.The subject is preferably human, but can also be a mammal in need ofveterinary treatment, e.g. domestic animals, farm animals, andlaboratory animals. For example, the subject may be a subject diagnosedwith a benign or malignant tumor, a cancer or a hyperplasia. The subjectmay be a cancer patient who is receiving treatment modalities againstcancer or has undergone a regimen of treatment, e.g., chemotherapy,radiation and/or surgery. The subject may be a cancer patient whosecancer appears to be regressing.

As used herein, the phrase “expression” is used to refer to thetranscription of a gene product into mRNA (gene expression) and is alsoused to refer to the expression of the protein encoded by the gene.

As used herein the term “over-expression” is used to refer to increasedproduction of a specific mRNA and/or protein in a cell, wherein theactual mRNA and protein product do not contain activating mutations. Asused herein, the term “over-activation”, as used to refer to Ras or anupstream or downstream effector, is used to refer to increased signalingthrough an otherwise non-activated form of a pathway member.Over-activation of a molecule typically results from increasedactivation (e.g. upstream signaling) or decreased de-activation (e.g.downstream negative regulation) of the molecule. Over-activation andover-expression of a specific gene/protein can co-exist, and often theexistence of one contributes to the existence of the other in a cell.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.Furthermore, therapeutically effective amounts will vary, as recognizedby those skilled in the art, depending on the specific disease treated,the route of administration, the excipient selected, and the possibilityof combination therapy. A physiological effect of a compound asdisclosed herein on the subject can be measured to determine thetherapeutically effective amount includes, without limitation, decreasedproliferation in a subject and the like.

It will be appreciated that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas of thisinvention, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. For purposes of this invention, heteroatoms such as nitrogenmay have hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. Furthermore, this invention is not intended to be limitedin any manner by the permissible substituents of organic compounds.

Combinations of substituents and variables envisioned by this inventionare preferably those that result in the formation of stable compoundsuseful in the treatment, for example of proliferative disorders,including, but not limited to cancer.

The term “stable”, as used herein, preferably refers to compounds whichpossess stability sufficient to allow manufacture and which maintain theintegrity of the compound for a sufficient period of time to be detectedand preferably for a sufficient period of time to be useful for thepurposes detailed herein.

The term “aliphatic”, as used herein, includes both saturated andunsaturated, straight chain (i.e., unbranched) or branched aliphatichydrocarbons, which are optionally substituted with one or morefunctional groups. As will be appreciated by one of ordinary skill inthe art, “aliphatic” is intended herein to include, but is not limitedto, alkyl, alkenyl, and alkynyl moieties. Thus, as used herein, the term“alkyl” includes straight and branched alkyl groups. An analogousconvention applies to other generic terms such as “alkenyl”, “alkynyl”,and the like. Furthermore, as used herein, the terms “alkyl”, “alkenyl”,“alkynyl”, and the like encompass both substituted and unsubstitutedgroups. In certain embodiments, as used herein, “lower alkyl” is used toindicate those alkyl groups (substituted, unsubstituted, branched orunbranched) having 1-6 carbon atoms.

The term “alkyl” is a saturated hydrocarbon in a molecule that is bondedto one other group in the molecule through a single covalent bond fromone of its carbon atoms. Alkyl groups can be cyclic or acyclic, branchedor unbranched (straight chained) and substituted or unsubstituted whenstraight chained or branched. An alkyl group typically has from 1 toabout 12 carbon atoms, for example, one to about six carbon atoms or oneto about four carbon atoms. Lower alkyl groups have one to four carbonatoms and include methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl and tert-butyl. When cyclic, an alkyl group typically containsfrom about 3 to about 10 carbons, for example, from about 3 to about 8carbon atoms, e.g., a cyclopropyl group, a cyclobutyl group, acyclopentyl group, a cyclohexyl group, a cycloheptyl group or acyclooctyl group.

In certain embodiments, the alkyl, alkenyl, and alkynyl groups employedin the invention contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-10 aliphatic carbon atoms.

In yet other embodiments, the alkyl, alkenyl, and alkynyl groupsemployed in the invention contain 1-8 aliphatic carbon atoms. In stillother embodiments, the alkyl, alkenyl, and alkynyl groups employed inthe invention contain 1-6 aliphatic carbon atoms.

In yet other embodiments, the alkyl, alkenyl, and alkynyl groupsemployed in the invention contain 14 carbon atoms. Illustrativealiphatic groups thus include, but are not limited to, for example,methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl,tert-butyl, n-pentyl, secpentyl, isopentyl, tert-pentyl, n-hexyl,sec-hexyl, moieties, and the like, which again, may bear one or moresubstituents. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike. Representative alkynyl groups include, but are not limited to,ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

The term “alicyclic”, as used herein, refers to compounds which combinethe properties of aliphatic and cyclic compounds and include but are notlimited to cyclic, or polycyclic aliphatic hydrocarbons and bridgedcycloalkyl compounds, which are optionally substituted with one or morefunctional groups. As will be appreciated by one of ordinary skill inthe art, “alicyclic” is intended herein to include, but is not limitedto, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties, which areoptionally substituted with one or more functional groups. Illustrativealicyclic groups thus include, but are not limited to, for example,cyclopropyl, —CH₂-cyclopropyl, cyclobutyl, —CH₂-cyclobutyl, cyclopentyl,—CH₂-cyclopentyl, cyclohexyl, —CH₂-cyclohexyl, cyclohexenylethyl,cyclohexanylethyl, norborbyl moieties, and the like, which may bear oneor more substituents.

The term “alkoxy” or “alkyloxyl” or “thioalkyl”, as used herein, refersto an alkyl group, as previously defined, attached to the parentmolecular moiety through an oxygen atom or through a sulfur atom. Incertain embodiments, the alkyl group contains 1-20 aliphatic carbonatoms. In certain other embodiments, the alkyl group contains 1-10aliphatic carbon atoms. In yet other embodiments, the alkyl groupcontains 1-8 aliphatic carbon atoms. In still other embodiments, thealkyl group contains 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl group contains 1-4 aliphatic carbon atoms.Examples of alkoxy, include, but are not limited to, methoxy, ethoxy,propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy.Examples of thioalkyl include, but are not limited to, methylthio,ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

The term “alkylamino” refers to a group having the structure —NHR′wherein R′ is alkyl, as defined herein. The term “aminoalkyl” refers toa group having the structure NH₂R′—, wherein R′ is alkyl, as definedherein. In certain embodiments, the alkyl group contains 1-20 aliphaticcarbon atoms. In certain other embodiments, the alkyl group contains1-10 aliphatic carbon atoms. In yet other embodiments, the alkylcontains 1-8 aliphatic carbon atoms. In still other embodiments, thealkyl group contains 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl group contains 1-4 aliphatic carbon atoms.Examples of alkylamino include, but are not limited to, methylamino,ethylamino, iso-propylamino, n-propylamino, and the like.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to, aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br, —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)Rx; —CO₂(Rx); —CON(Rx)₂; —OC(O)Rx; —OCO₂Rx; —OCON(Rx)₂;—N(Rx)₂; —S(O)₂Rx; —NRx(CO)Rx; wherein each occurrence of Rxindependently includes, but is not limited to, aliphatic, alycyclic,heteroaliphatic, heterocyclic, aryl, heteroaryl, alkylaryl, oralkylheteroaryl, wherein any of the aliphatic, heteroaliphatic,alkylaryl, or alkylheteroaryl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substituents are illustratedby the specific embodiments described herein.

In general, the term “aromatic moiety”, as used herein, refers to astable mono- or polycyclic, unsaturated moiety having preferably 3-14carbon atoms, each of which may be substituted or unsubstituted. Incertain embodiments, the term “aromatic moiety” refers to a planar ringhaving p-orbitals perpendicular to the plane of the ring at each ringatom and satisfying the Huckel rule where the number of pi electrons inthe ring is (4n+2), wherein n is an integer. A mono- or polycyclic,unsaturated moiety that does not satisfy one or all of these criteriafor aromaticity is defined herein as “non-aromatic,” and is encompassedby the term “alicyclic.” In general, the term “heteroaromatic moiety”,as used herein, refers to a stable mono- or polycyclic, unsaturatedmoiety having preferably 3-14 carbon atoms, each of which may besubstituted or unsubstituted; and comprising at least one heteroatomselected from O, S, and N within the ring (i.e., in place of a ringcarbon atom). In certain embodiments, the term “heteroaromatic moiety”refers to a planar ring comprising at least on heteroatom, havingp-orbitals perpendicular to the plane of the ring at each ring atom, andsatisfying the Huckel rule where the number of pi electrons in the ringis (4n+2), wherein n is an integer.

It will also be appreciated that aromatic and heteroaromatic moieties,as defined herein may be attached via an alkyl or heteroalkyl moiety andthus also include 5-(alkyl)aromatic, -(heteroalkyl)aromatic,-(heteroalkyl)heteroaromatic, and -(heteroalkyl)heteroaromatic moieties.Thus, as used herein, the phrases “aromatic or heteroaromatic moieties”and “aromatic, heteroaromatic, -(alkyl)aromatic, -(heteroalkyl)aromatic,-(heteroalkyl)heteroaromatic, and -(heteroalkyl)heteroaromatic” areinterchangeable. Substituents include, but are not limited to, any ofthe previously mentioned substituents, i.e., the substituents recitedfor aliphatic moieties, or for other moieties as disclosed herein,resulting in the formation of a stable compound.

The term “aryl”, as used herein, does not differ significantly from thecommon meaning of the term in the art, and refers to an unsaturatedcyclic moiety comprising at least one aromatic ring. In certainembodiments, “aryl” refers to a mono- or bicyclic carbocyclic ringsystem having one or two aromatic rings including, but not limited to,phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.

The term “heteroaryl”, as used herein, does not differ significantlyfrom the common meaning of the term in the art, and refers to a cyclicaromatic radical having from five to ten ring atoms of which one ringatom is selected from S, O, and N; zero, one, or two ring atoms areadditional heteroatoms independently selected from S, O, and N; and theremaining ring atoms are carbon, the radical being joined to the rest ofthe molecule via any of the ring atoms, such as, for example, pyridyl,pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl,oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,quinolinyl, isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups (includingbicyclic aryl groups) can be unsubstituted or substituted, whereinsubstitution includes replacement of one or more of the hydrogen atomsthereon independently with any one or more of the following moietiesincluding, but not limited to aliphatic; alicyclic; heteroaliphatic;heterocyclic; aromatic; heteroaromatic; aryl; heteroaryl; alkylaryl;heteroalkylaryl; alkylheteroaryl; heteroalkylheteroaryl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; —F; —Cl; —Br, —I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)Rx;—CO₂(Rx); —CON(Rx)₂; —OC(O)Rx; —OCO₂Rx; —OCON(Rx)₂; —N(Rx)₂; —S(O)₂Rx;and —NRx(CO)Rx; wherein each occurrence of Rx independently includes,but is not limited to, aliphatic, alicyclic, heteroaliphatic,heterocyclic, aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl,alkylheteroaryl, heteroalkylaryl or heteroalkylheteroaryl, wherein anyof the aliphatic, alicyclic, heteroaliphatic, heterocyclic, alkylaryl,or alkylheteroaryl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, saturated orunsaturated, and wherein any of the aromatic, heteroaromatic, aryl,heteroaryl, -(alkyl)aryl or (alkyl)heteroaryl substituents describedabove and herein may be substituted or unsubstituted. Additionally, itwill be appreciated, that any two adjacent groups taken together mayrepresent a 4, 5, 6, or 7-membered substituted or unsubstitutedalicyclic or heterocyclic moiety. Additional examples of generallyapplicable substituents are illustrated by the specific embodimentsdescribed herein.

The term “cycloalkyl”, as used herein, refers specifically to groupshaving three to ten, preferably three to seven carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and the like, which, as in thecase of aliphatic, alicyclic, heteroaliphatic or heterocyclic moieties,may optionally be substituted with substituents including, but notlimited to aliphatic; alicyclic; heteroaliphatic; heterocyclic;aromatic; heteroaromatic; aryl; heteroaryl; alkylaryl; heteroalkylaryl;alkylheteroaryl; heteroalkylheteroaryl; alkoxy; aryloxy; heteroalkoxy;heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F;—Cl; —Br, —I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(O)Rx; —CO₂(Rx); —CON(Rx)₂; —OC(O)Rx; —OCO₂Rx;—OCON(Rx)₂; —N(Rx)₂; —S(O)₂Rx; —NRx(CO)Rx; wherein each occurrence of Rxindependently includes, but is not limited to, aliphatic, alicyclic,heteroaliphatic, heterocyclic, aromatic, heteroaromatic, aryl,heteroaryl, alkylaryl, alkylheteroaryl, heteroalkylaryl orheteroalkylheteroaryl, wherein any of the aliphatic, alicyclic,heteroaliphatic, heterocyclic, alkylaryl, or alkylheteroarylsubstituents described above and herein may be substituted orunsubstituted, branched or unbranched, saturated or unsaturated, andwherein any of the aromatic, heteroaromatic, aryl or heteroarylsubstituents described above and herein may be substituted orunsubstituted. Additional examples of generally applicable substituentsare illustrated by the specific embodiments described herein.

The term “heteroaliphatic”, as used herein, refers to aliphatic moietiesin which one or more carbon atoms in the main chain have beensubstituted with a heteroatom. Thus, a heteroaliphatic group refers toan aliphatic chain which contains one or more oxygen, sulfur, nitrogen,phosphorus or silicon atoms, e.g., in place of carbon atoms.Heteroaliphatic moieties may be linear or branched, and saturated orunsaturated. In certain embodiments, heteroaliphatic moieties aresubstituted by independent replacement of one or more of the hydrogenatoms thereon with one or more moieties including, but not limited to,aliphatic; alicyclic; heteroaliphatic; heterocyclic; aromatic;heteroaromatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; —F; —Cl; —Br, —I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)Rx;—CO₂(Rx); —CON(Rx)₂; —OC(O)Rx; —OCO₂Rx; —OCON(Rx)₂; —N(Rx)₂; —S(O)₂Rx;and —NRx(CO)Rx; wherein each occurrence of Rx independently includes,but is not limited to, aliphatic, alicyclic, heteroaliphatic,heterocyclic, aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl,alkylheteroaryl, heteroalkylaryl or heteroalkylheteroaryl, wherein anyof the aliphatic, alicyclic, heteroaliphatic, heterocyclic, alkylaryl,or alkylheteroaryl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, saturated orunsaturated, and wherein any of the aromatic, heteroaromatic, aryl orheteroaryl substituents described herein may be substituted orunsubstituted. Additional examples of generally applicable substituentsare illustrated by the specific embodiments described herein.

The term “heterocycloalkyl”, “heterocycle” or “heterocyclic”, as usedherein, refers to compounds which combine the properties ofheteroaliphatic and cyclic compounds and include, but are not limitedto, saturated and unsaturated mono- or polycyclic cyclic ring systemshaving 5-16 atoms wherein at least one ring atom is a heteroatomselected from O, S, and N (wherein the nitrogen and sulfur heteroatomsmay be optionally be oxidized), wherein the ring systems are optionallysubstituted with one or more functional groups, as defined herein. Incertain embodiments, the term “heterocycloalkyl”, “heterocycle” or“heterocyclic” refers to a non-aromatic 5-, 6-, or 7-membered ring or apolycyclic group wherein at least one ring atom is a heteroatom selectedfrom 0, S, and N (wherein the nitrogen and sulfur heteroatoms may beoptionally be oxidized), including, but not limited to, a hi- ortri-cyclic group, comprising fused six-membered rings having between oneand three heteroatoms independently selected from oxygen, sulfur andnitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each6-membered ring has 0 to 2 double bonds and each 7-membered ring has 0to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may beoptionally be oxidized, (iii) the nitrogen heteroatom may optionally bequaternized, and (iv) any of the above heterocyclic rings may be fusedto an aryl or heteroaryl ring. Representative heterocycles include, butare not limited to, heterocycles such as furanyl, thiofuranyl, pyranyl,pyrrolyl, thienyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolyl,oxazolidinyl, isooxazolyl, isoxazolidinyl, dioxazolyl, thiadiazolyl,oxadiazolyl, tetrazolyl, triazolyl, thiatriazolyl, oxatriazolyl,thiadiazolyl, oxadiazolyl, morpholinyl, thiazolyl, thiazolidinyl,isothiazolyl, isothiazolidinyl, dithiazolyl, dithiazolidinyl,tetrahydrofuryl, and benzofused derivatives thereof. In certainembodiments, a “substituted heterocycle, or heterocycloalkyl orheterocyclic” group is utilized and as used herein, refers to aheterocycle, or heterocycloalkyl or heterocyclic group, as definedabove, substituted by the independent replacement of one, two or threeof the hydrogen atoms thereon with but are not limited to aliphatic;alicyclic; heteroaliphatic; heterocyclic; aromatic; heteroaromatic;aryl; heteroaryl; alkylaryl; heteroalkylaryl; alkylheteroaryl;heteroalkylheteroaryl; alkoxy; aryloxy; heteroaryloxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br, I; —OH;NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃;—C(O)Rx; —CO₂(Rx); —CON(Rx)₂; —OC(O)Rx; —OCO₂Rx; —OCON(Rx)₂; —N(Rx)₂;—S(O)₂Rx; —NRx(CO)Rx; wherein each occurrence of Rx independentlyincludes, but is not limited to, aliphatic, alicyclic; heteroaliphatic,heterocyclic, aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl,alkylheteroaryl, heteroalkylaryl, or heteroalkylheteroaryl, wherein anyof the aliphatic, alicyclic, heteroaliphatic, heterocyclic, alkylaryl,or alkylheteroaryl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, saturated orunsaturated, and wherein any of the aromatic, heteroaromatic, aryl, orheteroaryl substituents described herein may be substituted orunsubstituted. Additional examples or generally applicable substituentsare illustrated by the specific embodiments described herein.Additionally, it will be appreciated that any of the alicyclic orheterocyclic moieties described herein may comprise an aryl orheteroaryl moiety fused thereto. Additional examples of generallyapplicable substituents are illustrated by the specific embodimentsdescribed herein. The term “acyl”, as used herein, refers to acarbonyl-containing functionality, e.g., —C(O)R, wherein R is analiphatic, alycyclic, heteroaliphatic, heterocyclic, aryl, heteroaryl,(aliphatic)aryl, (heteroaliphatic)aryl, heteroaliphatic(aryl), orheteroaliphatic(heteroaryl) moiety, whereby each of the aliphatic,heteroaliphatic, aryl, or heteroaryl moieties is substituted orunsubstituted, or is a substituted (e.g., hydrogen or aliphatic,heteroaliphatic, aryl, or heteroaryl moieties) oxygen or nitrogencontaining functionality (e.g., forming a carboxylic acid, ester, oramide functionality). Acyl groups can be hydrolyzed or cleaved from acompound by enzymes, acids, or bases. One or more of the hydrogen atomsof an acyl group can be substituted, as described below. Typically, anacyl group is removed before a compound of the present invention bindsto a metal ion such as iron (III). Suitable substituents for alkyl andacyl groups include —OH, —O(R″), —COOH, ═O, —NH₂, —NH(R″), —NO₂,—COO(R″), —CONH₂, —CONH(R″), —CON(R″)₂, and guanidine. Each R″ isindependently an alkyl group or an aryl group. These groups canadditionally be substituted by an aryl group (e.g., an alkyl group canbe substituted with an aromatic group to form an arylalkyl group). Asubstituted alkyl or acyl group can have more than one substituent. Arylgroups include carbocyclic aromatic groups such as phenyl, p-tolyl,1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl. Aryl groups alsoinclude heteroaromatic groups such as N-imidazolyl, 2-imidazolyl,2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyranyl, 3-pyranyl, 3-pyrazolyl,4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-oxazolyl, 4-oxazolyl and 5-oxazolyl. Aryl groups alsoinclude fused polycyclic aromatic ring systems in which a carbocyclic,alicyclic, or aromatic ring or heteroaryl ring is fused to one or moreother heteroaryl or aryl rings. Examples include 2-benzothienyl,3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl, 2-indolyl, 3-indolyl,2-quinolinyl, 3-quinolinyl, 2-benzothiazolyl, 2-benzoxazolyl,2-benzimidazolyl, 1-isoquinolinyl, 3-isoquinolinyl, 1-isoindolyl and3-isoindolyl.

The term “O-protecting group” means a substituent which protectshydroxyl groups against undesirable reactions during syntheticprocedures. Examples of O-protecting groups include, but are not limitedto, methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl,2-(trimethylsilyl)ethoxymethyl, benzyl, triphenylmethyl,2,2,2-trichloroethyl, t-butyl, trimethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, methylene acetal, acetonide benzylidene acetal,cyclic ortho esters, methoxymethylene, cyclic carbonates, and cyclicboronates.

The term “leaving group” refers to a molecular fragment that can departswith a pair of electrons in heterolytic bond cleavage. Examples ofleaving groups include, but are not limited to, halides, such as F, Br,Cl, I; sulfonates, such as tosylates, nosylates, myselates; nonaflates;triflates; fluorosulfonates; nitrates; and phosphates.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine, and iodine.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.In certain embodiments, the alkyl group is perhalogenated (e.g.,perfluorinated).

The term “amino”, as used herein, refers to a primary (—NH₂), secondary(—NHRx), tertiary (—NRxRy), or quaternary (—N⁺RxRyRz) amine, where Rx,Ry, and Rz are independently an aliphatic, alicyclic, heteroaliphatic,heterocyclic, aromatic or heteroaromatic moiety, as defined herein.Examples of amino groups include, but are not limited to, methylamino,dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl,methylethylamino, isopropylamino, piperidino, trimethylamino, andpropylamino.

The term “alkylidene”, as used herein, refers to a substituted orunsubstituted, linear or branched saturated divalent radical of carbonand hydrogen atoms, having from one to n carbon atoms and having a freevalence at both ends of the radical. The alkylidene moiety may besubstituted.

The term “alkenylidene”, as used herein, refers to a substituted orunsubstituted, linear or branched unsaturated divalent radical of carbonand hydrogen atoms, having from two to n carbon atoms and having a freevalence at both ends of the radical, and wherein the unsaturation ispresent only as double bonds and wherein a double bond can exist betweenthe first carbon of the chain and the rest of the molecule. Thealkenylidene moiety may be substituted.

The term “alkynylidene”, as used herein, refers to a substituted orunsubstituted, linear or branched unsaturated divalent radical of carbonand hydrogen atoms, having from two to n carbon atoms, having a freevalence “-” at both ends of the radical, and wherein the unsaturation ispresent only as triple bonds and wherein a triple bond can exist betweenthe first carbon of the chain and the rest of the molecule. Thealkynylidene moiety may be substituted.

The term “carbamate”, as used herein, refers to any carbamate derivativeknown to one of ordinary skill in the art. Examples of carbamatesinclude t-Boc, Fmoc, benzyloxycarbonyl, alloc, methyl carbamate, ethylcarbamate, 9-(2-sulfo)fluorenylmethyl carbamate,9-(2,7-dibromo)fluorenylmethyl carbamate, Tbfmoc, Climoc, Bimoc,DBD-Tmoc, Bsmoc, Troc, Teoc, 2-phenylethyl carbamate, Adpoc,2-chloroethyl carbamate, 1,1-dimethyl-2-haloethyl carbamate, DB-t-BOC,TCBOC, Bpoc, t-Bumeoc, Pyoc, Bnpeoc,N-(2-pivaloylamino)-1,1-dimethylethyl carbamate, NpSSPeoc. In certainembodiments, carbamates are used as nitrogen protecting groups.

Unless otherwise indicated, as used herein, the terms “alkyl”,“alkenyl”, “alkynyl”, “heteroalkyl”,“heteroalkenyl”, “heteroalkynyl”,“alkylidene”, “alkynylidene”, -(alkyl)aryl, -(heteroalkyl)aryl,-(heteroalkyl)aryl, -(heteroalkyl)heteroaryl, and the like encompasssubstituted and unsubstituted, and linear and branched groups.Similarly, the terms “aliphatic”, “heteroaliphatic”, and the likeencompass substituted and unsubstituted, saturated and unsaturated, andlinear and branched groups. Similarly, the terms “cycloalkyl”,“heterocycle”, “heterocyclic”, and the like encompass substituted andunsubstituted, and saturated and unsaturated groups. Additionally, theterms “cycloalkenyl”, “cycloalkynyl”, “heterocycloalkenyl”,“heterocycloalkynyl”, “aromatic”, “heteroaromatic, “aryl”, “heteroaryl”,and the like encompass both substituted and unsubstituted groups.

Acids commonly employed to form acid addition salts from compounds withbasic groups are inorganic acids such as hydrochloric acid, hydrobromicacid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, andorganic acids such as p-toluenesulfonic acid, methanesulfonic acid,oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid,citric acid, benzoic acid, acetic acid, and the like. Examples of suchsalts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

The phrase, “pharmaceutically acceptable derivative”, as used herein,denotes any pharmaceutically acceptable salt, ester, or salt of suchester, of such compound, or any other adduct or derivative which, uponadministration to a patient, is capable of providing (directly orindirectly) a compound as otherwise described herein, or a metabolite orresidue thereof. Pharmaceutically acceptable derivatives thus includeamong others pro-drugs. A pro-drug is a derivative of a compound,usually with significantly reduced pharmacological activity, whichcontains an additional moiety, which is susceptible to removal in vivoyielding the parent molecule as the pharmacologically active species. Anexample of a pro-drug is an ester, which is cleaved in vivo to yield acompound of interest. Pro-drugs of a variety of compounds, and materialsand methods for derivatizing the parent compounds to create thepro-drugs, are known and may be adapted to the present invention. Thebiological activity of pro-drugs may also be altered by appendingfunctionality onto the compound, which may be catalyzed by an enzyme.Also, included are oxidation and reduction reactions, includingenzyme-catalyzed oxidation and reduction reactions. Certain exemplarypharmaceutical compositions and pharmaceutically acceptable derivativesare discussed in more detail herein.

“Compound”: The term “compound” or “chemical compound” as used hereincan include organometallic compounds, organic compounds, metals,transitional metal complexes, and small molecules. In certainembodiments, polynucleotides are excluded from the definition ofcompounds. In other embodiments, polynucleotides and peptides areexcluded from the definition of compounds. In certain embodiments, theterm compound refers to small molecules (e.g., preferably, non-peptidicand non-oligomeric) and excludes peptides, polynucleotides, transitionmetal complexes, metals, and organometallic compounds.

“Small Molecule”: As used herein, the term “small molecule” refers to anon-peptidic, non-oligomeric organic compound, either synthesized in thelaboratory or found in nature. A small molecule is typicallycharacterized in that it contains several carbon-carbon bonds, and has amolecular weight of less than 2000 g/mol, preferably less than 1500g/mol, although this characterization is not intended to be limiting forthe purposes of the 5 present invention. Examples of “small molecules”that occur in nature include, but are not limited to, taxol, dynemicityand rapamycin, Examples of “small molecules” that are synthesized in thelaboratory include, but are not limited to, compounds described in Tanet al., (“Stereoselective Synthesis of over Two Million Compounds HavingStructural Features Both Reminiscent of Natural Products and Compatiblewith Miniaturized Cell-Based Assays” J. Am. Chem. Soc. 1998, 120, 8565;incorporated herein by reference).

“Biological sample”: As used herein the term “biological sample”includes, without limitation, cell cultures, or extracts thereof;biopsied material obtained from an animal (e.g., mammal) or extractsthereof; and blood, saliva, urine, feces, semen, tears, or other bodyfluids or extracts thereof. For example, the term “biological sample”refers to any solid or fluid sample obtained from, excreted by orsecreted by any living organism, including single-celled micro-organisms(such as bacteria and yeasts) and multicellular organisms (such asplants and animals, for instance a vertebrate or a mammal, and inparticular a healthy or apparently healthy human subject or a humanpatient affected by a condition or disease to be diagnosed orinvestigated). The biological sample can be in any form, including asolid material such as a tissue, cells, a cell pellet, a cell extract,cell homogenates, or cell fractions; or a biopsy, or a biological fluid.The biological fluid may be obtained from any site (e.g., blood, saliva(or a mouth wash containing buccal cells), tears, plasma, serum, urine,bile, cerebrospinal fluid, amniotic fluid, peritoneal fluid, and pleuralfluid, or cells therefrom, aqueous or vitreous humor, or any bodilysecretion), a transudate, an exudate (e.g., fluid obtained from anabscess or any other site of infection or inflammation), or fluidobtained from a joint (e.g., a normal joint or a joint affected bydisease such as rheumatoid arthritis, osteoarthritis, gout or septicarthritis). The biological sample can be obtained from any organ ortissue (including a biopsy or autopsy specimen) or may comprise cells(whether primary cells or cultured cells) or medium conditioned by anycell, tissue, or organ. Biological samples may also include sections oftissues such as frozen sections taken for histological purposes.

Biological samples also include mixtures of biological moleculesincluding proteins, lipids, carbohydrates, and nucleic acids generatedby partial or complete fractionation of cell or tissue homogenates.Although the sample is preferably taken from a human subject, biologicalsamples may be from any animal, plant, bacteria, virus, yeast, etc.

“Pharmaceutically acceptable salt”: As used herein, the term“pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response, and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts ofamines, carboxylic acids, and other types of compounds, are well knownin the art. For example, Berge et al. describe pharmaceuticallyacceptable salts in detail in J. Pharmaceutical Sciences 1977, 6, 1-19,incorporated herein by reference. The salts can be prepared in situduring the final isolation and purification of a compound of theinvention, or separately by reacting a free base or free acid functionwith a suitable reagent, as described generally below. For example, afree base can be reacted with a suitable acid. Furthermore, where thecompound of the invention carries an acidic moiety, suitablepharmaceutically acceptable salts thereof may, include metal salts suchas alkali metal salts, e.g. sodium or potassium salts; and alkalineearth metal salts, e.g. calcium or magnesium salts. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid; or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptoate,hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate,laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate, and aryl sulfonate.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level (e.g., in the absenceof a compound of the invention).

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statistically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level (e.g., in the absence of a compound of the invention).

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker. The term refersto statistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true. The decision is often made using thep-value.

The term “substantially” as used herein means a proportion of at leastabout 60%, or preferably at least about 70%, or at least about 80%, orat least about 90%, or at least about 95%, or at least about 97% or atleast about 99% or more, or any integer between 70% and 100%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

In this application and the claims, the use of the singular includes theplural unless specifically stated otherwise. In addition, use of “or”means “and/or” unless stated otherwise. Moreover, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one unit unless specifically statedotherwise.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology, andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 18th Edition, published by Merck Research Laboratories, 2006(ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006. Definitions of common terms inmolecular biology are found in Benjamin Lewin, Genes IX, published byJones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew etal. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982);Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989);Davis et al., Basic Methods in Molecular Biology, Elsevier SciencePublishing, Inc., New York, USA (1986); or Methods in Enzymology: Guideto Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. KimmerlEds., Academic Press Inc., San Diego, USA (1987); Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et al., ed., John Wiley and Sons, Inc.) and Current Protocolsin Immunology (CPI) (John E. Coligan, et al., ed. John Wiley and Sons,Inc.), which are all incorporated by reference herein in theirentireties.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose of skill in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications, and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddo not constitute any admission as to the correctness of the dates orcontents of these documents.

Compounds of the Invention

Compounds of this invention include those, as set forth above anddescribed herein, and are illustrated in part by the various classes,subgenera, and species disclosed elsewhere herein. The present inventionprovides compounds that inhibit PKC delta, having the general formula(Ia), (IIa), (IIIa), (IVa), or (V), a set forth in FIGS. 11 A-M and 12A-F inclusive.

In certain embodiments, the compound is of formula (Ia) wherein Z isCH₂, O, NH, S, C(R′″)(R′″); each occurrence of R′ is independentlyhydrogen; halogen; cyclic or acyclic, substituted or unsubstituted,branched or unbranched aliphatic; cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic; substituted orunsubstituted, branched or unbranched acyl; substituted orunsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; ORB; —C(═O)RB; —CO₂RB;—C(═O)N(RB)₂; —CN; —SCN; —SRB; —SORB; —SO₂RB₂0; —NO₂; —N(RB)₂;—NHC(O)RB; or —C(RB)₃; wherein each occurrence of RB is independentlyhydrogen; halogen; a protecting group; aliphatic; heteroaliphatic; acyl;aryl moiety; heteroaryl; hydroxyl; alkoxy; aryloxy; alkylthioxy;arylthioxy; amino; alkylamino; dialkylamino; heteroaryloxy; orheteroarylthioxy; two R′ can be taken together to form a fused cyclicgroup; each occurrence of R″ is independently hydrogen, halogen; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedaliphatic; cyclic or acyclic, substituted or unsubstituted, branched orunbranched heteroaliphatic; substituted or unsubstituted, branched orunbranched acyl; substituted or unsubstituted, branched or unbranchedaryl; substituted or unsubstituted, branched or unbranched heteroaryl;ORB; —C(═O)RB; —CO₂RB; —C(═O)N(RB)₂; —CN; —SCN; —SRB; —SORB; —SO₂RB;—NO₂; —N(RB)₂; —NHC(O)RB₃; or —C(RB)₃; wherein each occurrence of RB isindependently hydrogen; halogen; a protecting group; aliphatic;heteroaliphatic; acyl; aryl moiety; heteroaryl; hydroxyl; alkoxy;aryloxy; alkylthioxy; arylthioxy; amino; alkylamino; dialkylamino;heteroaryloxy; or heteroarylthioxy; each occurrence of R′″ isindependently hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; ORB; —C(═O)RB; —CO₂RB;—C(═O)N(RB)₂; —CN; —SCN; —SRB; —SORB; —SO₂RB; —NO₂; —N(RB)₂; —NHC(O)RB;or —C(RB)₃; wherein each occurrence of RB is independently hydrogen;halogen; a protecting group; aliphatic; heteroaliphatic; acyl; aryl;heteroaryl; hydroxyl; alkoxy; aryloxy; alkylthioxy; arylthioxy; amino;alkylamino; dialkylamino; heteroaryloxy; or heteroarylthioxy; and n isan integer 1-4, inclusive, and pharmaceutically acceptable saltsthereof.

In some embodiments, Z is CH2, O, NH, S, C(R′″)(R′″). In someembodiments, each occurrence of R′ is independently hydrogen; hydroxyl,ORB; wherein RB is hydrogen; a protecting group; C₁₋₆ alkyl; aryl; orheteroaryl.

In some embodiments, each occurrence of R″ is independently hydrogen;hydroxyl, or ORB; wherein RB is hydrogen; a protecting group; or C₁₋₆alkyl. In some embodiments, each occurrence of R′″ is independentlyhydrogen; C₁₋₆ alkyl hydroxyl, or ORB; wherein RB is hydrogen; aprotecting group; or C₁₋₆ alkyl. In some embodiments, n is an integer1-4, inclusive.

In certain embodiments, the compound is of formula (IIa) wherein Z isCH₂, O, NH, S, or C(R′″)(R′″); X is CH₂, or C(═O); Y is CH₂, or C(═O);each occurrence of R′ is independently hydrogen; halogen; cyclic oracyclic, substituted or unsubstituted, branched or unbranched aliphatic;cyclic or acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl; ORB;—C(═O)RB; —CO₂RB; —C(═O)N(RB)₂; —CN; —SCN; —SRB; —SORB; —SO₂RB; —NO₂;—N(RB)₂; —NHC(O)RB; or —C(RB)₃; wherein each occurrence of RB isindependently hydrogen; halogen; a protecting group; aliphatic;heteroaliphatic; acyl; aryl moiety; heteroaryl; hydroxyl; alkoxy;aryloxy; alkylthioxy; arylthioxy; amino; alkylamino; dialkylamino;heteroaryloxy; or heteroarylthioxy; two R′ can be taken together to forma fused cyclic group; each occurrence of R″ is independently hydrogen,halogen; cyclic or acyclic, substituted or unsubstituted branched orunbranched aliphatic; cyclic or acyclic, substituted or unsubstituted,branched or unbranched heteroaliphatic; substituted or unsubstituted,branched or unbranched acyl; substituted or unsubstituted, branched orunbranched aryl; substituted or unsubstituted, branched or unbranchedheteroaryl; ORB; —C(═O)RB; —CO₂RB; —C(═O)N(RB)₂; —CN; —SCN; —SRB; —SORB;—SO₂RB; —NO₂; —N(RB)₂; —NHC(O)RB; or —C(RB)₃; wherein each occurrence ofRB is independently hydrogen; halogen; a protecting group; aliphatic;heteroaliphatic; acyl; aryl moiety; heteroaryl; hydroxyl; alkoxy;aryloxy; alkylthioxy; arylthioxy; amino; alkylamino; dialkylamino;heteroaryloxy; or heteroarylthioxy; each occurrence of R′″ isindependently hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; ORB; —C(═O)RB; —CO₂RB;—C(═O)N(RB)₂; —CN; —SCN; —SRB; —SORB; —SO₂RB; —NO₂; —N(RB)₂; —NHC(O)RB;or —C(RB)₃; wherein each occurrence of RB is independently hydrogen;halogen; a protecting group; aliphatic; heteroaliphatic; acyl; aryl;heteroaryl; hydroxyl; alkoxy; aryloxy; alkylthioxy; arylthioxy; amino;alkylamino; dialkylamino; heteroaryloxy; or heteroarylthioxy; and n isan integer 1-4, inclusive, and pharmaceutically acceptable saltsthereof.

In some embodiments, Z is CH2, O, NH, S, C(R′″)(R′″). In someembodiments, X is CH₂, or C(═O). In some embodiments, Y is CH₂, orC(═O). In some embodiments, each occurrence of R′ is independentlyhydrogen; hydroxyl, ORB; wherein RB is hydrogen; a protecting group;C₁₋₆ alkyl; aryl; or heteroaryl. In some embodiments, each occurrence ofR″ is independently hydrogen; hydroxyl, or ORB; wherein RB is hydrogen;a protecting group; or C₁₋₆ alkyl. In some embodiments each occurrenceof R′″ is independently hydrogen; C₁₋₆ alkyl hydroxyl, or ORB; whereinRB is hydrogen; a protecting group; or C₁₋₆ alkyl. In some embodiments,n is an integer 1-4, inclusive.

In certain embodiments, the compound is of formula (IIIa) wherein eachoccurrence of R′ is independently hydrogen; halogen; cyclic or acyclic,substituted or unsubstituted, branched or unbranched aliphatic; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl; —ORB;—C(═O)RB; —CO₂RB; —C(═O)N(RB)₂; —CN; —SCN; —SRB; —SORB; —SO₂RB; —NO₂;—N(RB)₂; —NHC(O)RB; or —C(RB)₃; wherein each occurrence of RB isindependently hydrogen; halogen; a protecting group; aliphatic;heteroaliphatic; acyl; aryl moiety; heteroaryl; hydroxyl; alkoxy;aryloxy; alkylthioxy; arylthioxy; amino; alkylamino; dialkylamino;heteroaryloxy; or heteroarylthioxyl, and pharmaceutically acceptablesalts thereof.

In some embodiments each occurrence of R′ is independently hydrogen;hydroxyl, ORB; wherein RB is hydrogen; a protecting group; C₁₋₆ alkyl;aryl; or heteroaryl.

In certain embodiment, the compound is of formula (IVa) wherein X isCH₂, or C(═O); Y is CH₂, or C(═O); each occurrence of R′″ eachoccurrence of R″ is independently hydrogen; C₁₋₆ alkyl hydroxyl, or ORB;wherein RB is hydrogen; a protecting group; or C₁₋₆ alkyl; andpharmaceutically acceptable salts thereof.

In certain embodiments, the compound is of formula (V) are as disclosedin FIGS. 12C and 12D. In some embodiments, the compound of formula (Ia),(IIa), (IIIa), (IVa), or (V) as disclosed herein does not include any ofN- -phenethyl-carbazolen (SMILES: C(Cn1c2ccccc2c2ccccc12)c1ccccc1),crotmadine (SMILES: CC1(C)CCc2c(O)ccc(C(═O)\C═C\c3ccc(O)cc3)c201),9-(1H-inden-2-yl)-9H-carbazole (SMILES:C1C(═Cc2ccccc12)n1c2ccccc2c2ccccc12), rottlerin (mallatoxin), NDGAderivative tetra-a-methyl nordihydroguaiaric acid (M4N or terameprocol),UNC-01(7-0H staurosporine) and CGP41251 (PKC412, 4′-N-benzoylstaurosporine), KAI-9803.

Pharmaceutical Compositions

Aspects of the present invention relate to the use of any or, or acombination of compounds of formula (Ia), (IIa), (IIIa), (IVa), or (V)as disclosed herein as an inhibitor of PKC delta. In particular, acompound of (Ia), (IIa), (IIIa), (IVa), or (V) as disclosed hereinselectively inhibits PKC delta over other PKC isoforms. In particular, acompound of formula (Ia), (IIa), (IIIa), (IVa), or (V) as disclosedherein as a selective PKC delta inhibitors are advantageous overexisting PKC delta inhibitors, as they are more potent at inhibitingcell proliferation and are non-toxic to cells with normal levels p21Rassignaling.

Additionally, while some PKC delta inhibitors are used in combination,for example, rottlerin and straurosporine, the present PKC deltainhibitor compounds of formula (Ia), (IIa), (IIIa), (IVa), or (V) areadvantageous at inhibiting PKC delta compounds in a single molecule.

The present invention provides novel compounds useful in the treatmentof diseases or disorders associated with PKC delta activity. Thecompounds are useful in the treatment of a disease or condition thatbenefit from inhibition of PKC delta, for example, in a disease orcondition where there is an elevation or increase in the activity and/orexpression of PKC delta. In some embodiments, the compounds as disclosedherein are useful for methods for the treatment or prevention ofdisorders where inhibition of PKC delta is desirable, for example,diseases associated with impaired insulin sensitivity or fatty liverdisease (FLD), including hepatic steatosis and type 2 diabetes, andnon-alcoholic steatohepatitis (NASH), according to the methods asdisclosed in International Application W0/2011/041385 which isincorporated herein in its entirety by reference.

In certain embodiments, the inventive compounds as disclosed herein areuseful in the treatment of proliferative diseases, such as cancer. Insome embodiments, the cancer is for example, a bronchopulmonary cancer,a gastrointestinal cancer or a pancreatic neuroendocrine cancer, acarcinoid and/or neuroendocrine cancer, malignant melanoma, pancreatic,gastrointestinal or lung cancer. In some embodiments, a neuroendocrinecancer can be derived from a bronchopulmonary, or foregut or hindguttumor. (e.g., neuroendocrine tumor of pulmonary and gastrointestinalorigin).

In certain embodiments, the inventive compounds as disclosed herein areuseful in the treatment of diseases characterized by proliferation ofconnective tissue cells, or excessive deposition of matrix by thosecells, such as in the fibrotic diseases. The fibrotic diseases encompassa wide spectrum of clinical entities, including multisystemic diseases,such as systemic sclerosis (scleroderma), multifocal fibrosclerosis,sclerodermatous graft-versus-host disease in bone marrow transplantrecipients, and nephrogenic systemic fibrosis, as well as organ-specificdisorders, such as pulmonary, liver, and kidney fibrosis. Specificallyincluded in these fibrotic diseases are: Chronic Kidney Disease, LiverFibrosis, Pulmonary/Lung Fibrosis, Systemic Sclerosis, Idiopathicpulmonary fibrosis; Cystic fibrosis, Cirrhosis, Endomyocardial fibrosis(heart); Mediastinal fibrosis (soft tissue of the mediastinum),Myelofibrosis (bone marrow), Retroperitoneal fibrosis (soft tissue ofthe retroperitoneum), Progressive massive fibrosis (lungs); acomplication of coal workers' pneumoconiosis, Nephrogenic systemicfibrosis (skin), Crohn's Disease (intestine), Keloid (skin), Oldmyocardial infarction (heart), Scleroderma/systemic sclerosis (skin,lungs), Arthrofibrosis (knee, shoulder, other joints), and some forms ofadhesive capsulitis (shoulder), and fibrosis following radiation(“post-radiation fibrosis”), and following administration of certaindrugs, such as bleomycin (“bleomycin-induced pulmonary fibrosis”).

Although their etiology and causative mechanisms differ, the fibroticdiseases share the common feature of disordered and exaggerateddeposition of extracellular matrix in affected tissues. Elevatedexpression of genes encoding extracellular matrix proteins is a commonand characteristic feature of these conditions, and the resultingfibrosis disrupts the normal architecture of the affected organs, whichultimately leads to their dysfunction and failure. The persistentactivation of fibroblastic cells distinguishes controlled repair, suchas that occurring during normal wound healing, from the uncontrolledfibrosis that is the hallmark of this group of diseases. Transforminggrowth factor-beta (TGF-beta) is a critical mediator in the pathogenesisof tissue fibrosis. One TGF-beta pathway involves protein kinase C-delta(PKC-delta). In response to TGF-beta, PKC delta is phosphorylated;phosphorylated PKC delta then removes inhibitory factors from thecollagen gene promoter in the nucleus, which increases thetranscriptional activity of the collagen gene. Inhibition of PKC-deltaby pharmacologic or molecular biological techniques diminished theincreased collagen gene expression induced by TGF-beta and that ofcultured systemic sclerosis fibroblasts (Jimenez S A, Gaidarova S,Saitta B, Sandorfi N, Herrich D J, Rosenbloom J C. et al., Role ofprotein kinase C-delta in the regulation of collagen gene expression inscleroderma fibroblasts. J Clin. Invest. 2001; 1081395-403).

Accordingly, in another aspect of the present invention, pharmaceuticalcompositions are provided, which comprise any one of the compoundsdescribed herein (or a prodrug, pharmaceutically acceptable salt orother pharmaceutically acceptable derivative thereof) and optionally apharmaceutically acceptable excipient. In certain embodiments, thesecompositions optionally further comprise one or more additionaltherapeutic agents. Alternatively, a compound of this invention may beadministered to a patient in need thereof in combination with theadministration of one or more other therapeutic agents. For example, inthe treatment of cancer, an additional therapeutic agent for conjointadministration or inclusion in a pharmaceutical composition with acompound of this invention may be an approved chemotherapeutic agent.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative thereof. According to thepresent invention, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or a pro-drug or other adduct or derivative of acompound of this invention which upon administration to a patient inneed is capable of providing, directly or indirectly, a compound asotherwise described herein, or a metabolite or residue thereof.

As described above, the pharmaceutical compositions of the presentinvention optionally comprise a pharmaceutically acceptable excipient,which, as used herein, includes any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives,antioxidants, solid binders, lubricants, and the like, as suited to theparticular dosage form desired. Remington's Pharmaceutical Sciences,Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980)discloses various excipients used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional excipient medium is incompatible with thecompounds of the invention, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable excipientsinclude, but are not limited to, sugars such as lactose, glucose, andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl cellulose,and cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients such as cocoa butter and suppository waxes; oils such aspeanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; cornoil and soybean oil; glycols; such as propylene glycol; esters such asethyl oleate and ethyllaurate; agar, cyclodextrins and derivatives,buffering agents such as magnesium hydroxide and aluminum hydroxide;alginic acid; pyrogen-free water; isotonic saline; Ringer's solution;ethyl alcohol, and phosphate buffer solutions, as well as othernon-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives, and antioxidants can also be present in the composition,according to the judgment of the formulator.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, com, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedia prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension orcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionthat, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude (poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol, or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or di-calcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monosteamte, h) absorbents such as kaolin andbentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols, andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols, and the like. The activecompounds can also be in micro-encapsulated form with one or moreexcipients as noted above. The solid dosage forms of tablets, dragees,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose and starch. Such dosage forms may alsocomprise, as in normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such asmagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

The present invention encompasses pharmaceutically acceptable topicalformulations of inventive compounds. The term “pharmaceuticallyacceptable topical formulation”, as used herein, means any formulationwhich is pharmaceutically acceptable for intradermal administration of acompound of the invention by application of the formulation to theepidermis. In certain embodiments of the invention, the topicalformulation comprises a excipient system.

Pharmaceutically effective excipients include, but are not limited to,solvents (e.g., alcohols, poly alcohols, water), creams, lotions,ointments, oils, plasters, liposomes, powders, emulsions,microemulsions, and buffered solutions (e.g., hypotonic or bufferedsaline) or any other excipient known in the art for topicallyadministering pharmaceuticals. A more complete listing of art-knowncarvers is provided by reference texts that are standard in the art, forexample, Remington's Pharmaceutical Sciences, 16th Edition, 1980 and17^(th) Edition, 1985, both published by Mack Publishing Company,Easton, Pa., the disclosures of which are incorporated herein byreference in their entireties. In certain other embodiments, the topicalformulations of the invention may comprise excipients. Anypharmaceutically acceptable excipient known in the art may be used toprepare the inventive pharmaceutically acceptable topical formulations.Examples of excipients that can be included in the topical formulationsof the invention include, but are not limited to, preservatives,antioxidants, moisturizers, emollients, buffering agents, solubilizingagents, other penetration agents, skin protectants, surfactants, andpropellants, and/or additional therapeutic agents used in combination tothe inventive compound. Suitable preservatives include, but are notlimited to, alcohols, quaternary amines, organic acids, parabens, andphenols. Suitable antioxidants include, but are not limited to, ascorbicacid and its esters, sodium bisulfite, butylated hydroxytoluene,butylated hydroxyarrisole, tocopherols, and chelating agents like EDTAand citric acid. Suitable moisturizers include, but are not limited to,glycerine, sorbitol, polyethylene glycols, urea, and propylene glycol.Suitable buffering agents for use with the invention include, but arenot limited to, citric, hydrochloric, and lactic acid buffers. Suitablesolubilizing agents include, but are not limited to, quaternary ammoniumchlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates.Suitable skin protectants that can be used in the topical formulationsof the invention include, but are not limited to, vitamin E oil,allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

In certain embodiments, the pharmaceutically acceptable topicalformulations of the invention comprise at least a compound of theinvention and a penetration enhancing agent. The choice of topicalformulation will depend or several factors, including the condition tobe treated, the physicochemical characteristics of the inventivecompound and other excipients present, their stability in theformulation, available manufacturing equipment, and costs constraints.As used herein the term “penetration enhancing agent” means an agentcapable of transporting a pharmacologically active compound through thestratum corneum and into the epidermis or dermis, preferably, withlittle or no systemic absorption. A wide variety of compounds have beenevaluated as to their effectiveness in enhancing the rate of penetrationof drugs through the skin. See, for example, Percutaneous PenetrationEnhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., BocaRaton, Fla. (1995), which surveys the use and testing of various skinpenetration enhancers, and Buyuktimkin et al., Chemical Means ofTransdermal Drug Permeation Enhancement in Transdermal and Topical DrugDelivery Systems, Gosh T. K., Pfister W. R., Yum S. I. (Eds.),Interpharm Press Inc., Buffalo Grove, Ill. (1997). In certain exemplaryembodiments, penetration agents for use with the invention include, butare not limited to, triglycerides (e.g., soybean oil), aloe compositions(e.g., aloevera gel), ethyl alcohol, isopropyl alcohol,octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400,propylene glycol, N-decylmethylsulfoxide, fatty acid esters (e.g.,isopropyl myristate, methyllaurate, glycerol monooleate, and propyleneglycol monooleate), and N-methyl pyrrolidone.

In certain embodiments, the compositions may be in the form ofointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants or patches. In certain exemplary embodiments, formulations ofthe compositions according to the invention are creams, which mayfurther contain saturated or unsaturated fatty acids such as stearicacid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleylalcohols, stearic acid being particularly preferred.

Creams of the invention may also contain a non-ionic surfactant, forexample, polyoxy-40-stearate. In certain embodiments, the activecomponent is admixed under sterile conditions with a pharmaceuticallyacceptable excipient and any needed preservatives or buffers as may berequired. Ophthalmic formulation, eardrops, and eye drops are alsocontemplated as being within the scope of this invention. Additionally,the present invention contemplates the use of transdermal patches, whichhave the added advantage of providing controlled delivery of a compoundto the body. Such dosage forms are made by dissolving or dispensing thecompound in the proper medium. As discussed above, penetration enhancingagents can also be used to increase the flux of the compound across theskin. The rate can be controlled by either providing a rate controllingmembrane or by dispersing the compound in a polymer matrix (e.g., PLGA)or gel.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be formulated and employed incombination therapies, that is, the compounds and pharmaceuticalcompositions can be formulated with or administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. It willalso be appreciated that the therapies employed may achieve a desiredeffect for the same disorder (for example, an inventive compound may beadministered concurrently with another immunomodulatory agent oranticancer agent), or they may achieve different effects (e.g., controlof any adverse effects).

For example, other therapies or anticancer agents that may be used incombination with the inventive compounds of the present invention forcancer therapy include surgery, radiotherapy (in but a few examples,y-radiation, neutron beam radiotherapy, electron beam radiotherapy,proton therapy, brachytherapy, and systemic radioactive isotopes, toname a few), endocrine therapy, biologic response modifiers (interferon,interleukins, and tumor necrosis factor (TNF) to name a few),hyperthermia and cryotherapy, agents to attenuate any adverse effects(e.g., antiemetics), and other approved chemotherapeutic drugs,including, but not limited to, alkylating drugs (mechlorethamine,chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites(Methotrexate), purine antagonists and pyrimidine antagonists(6-Mercaptopurine, 5-Fluorouracil, Cytarabile, Gemcitabine), spindlepoisons (Vinblastine, Vincristine, Vinorelbine, Paclitaxel),podophyllotoxins (Etoposide, Irinotecan, Topotecan), antibiotics(Doxorubicin, Bleomycin, Mitomycin), nitrosoureas (Carmustine,Lomustine), inorganic ion (Cisplatin, Carboplatin), enzymes(Asparaginase), and hormones (Tamoxifen, Leuprelide, Flutamide, andMegestrol), to name a few. For a more comprehensive discussion ofupdated cancer therapies see, The Merck Manual, Seventeenth Ed. 1999,the entire contents of which are hereby incorporated by reference. Seealso the National Cancer Institute (CNI) website and the Food and DrugAdministration (FDA) website for a list of the FDA approved oncologydrugs.

In certain embodiments, the pharmaceutical compositions of the presentinvention further comprise one or more additional therapeutically activeingredients (e.g., chemotherapeutic and/or palliative). For purposes ofthe invention, the term “palliative” refer, to treatment that is focusedon the relief of symptoms of a disease and/or side effects of atherapeutic regimen, but is not curative. For example, palliativetreatment encompasses painkillers, anti-nausea medication andanti-sickness drugs. In addition, chemotherapy, radiotherapy and surgerycan all be used palliatively (that is, to reduce symptoms without goingfor cure; e.g., for shrinking tumors and reducing pressure, bleeding,pain and other symptoms of cancer).

Additionally, the present invention provides pharmaceutically acceptablederivatives of the inventive compounds, and methods of treating asubject using these compounds, pharmaceutical compositions thereof, oreither of these in combination with one or more additional therapeuticagents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative thereof. According to thepresent invention, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or a prodrug or other adduct or derivative of a compoundof this invention which upon administration to a patient in need iscapable of providing, directly or indirectly, a compound as otherwisedescribed herein, or a metabolite or residue thereof.

Another aspect of the invention relates to a kit for conveniently andeffectively carrying out the methods in accordance with the presentinvention. In general, the pharmaceutical pack or kit comprises one ormore containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Such kits are especiallysuited for the topical delivery of the inventive compounds. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

Pharmaceutical Uses and Methods of Treatment

One aspect of the present invention relates to the use of the compoundsof formula (Ia), (IIa), (IIIa), (IVa), or (V) as disclosed herein in amethod to treat a disease or disorder associated with aberrant PKC deltaactivity. Accordingly, in some embodiments, the compounds are useful inthe treatment of a disease or condition that benefit from inhibition ofPKC delta, for example, in a disease or condition where there is anelevation or increase in the activity and/or expression of PKC delta. Ingeneral, methods of using the compounds of the present inventioncomprise administering to a subject in need thereof a therapeuticallyeffective amount of a compound of the present invention. The compoundsof the invention are generally inhibitors of PKC delta activity. Asdiscussed above, the compounds of the invention are typically inhibitorsof PKC delta and, as such, are useful in the treatment of disorderswhere PKC delta is increased or overexpressed. Diseases associated withPKC delta may be treated by an inventive compound that inhibits PKCdelta.

PKC Delta

Without wishing to be bound by theory, there are at least 12 PKCisoforms that are classified into three subfamilies according to thestructure of the N-terminal regulatory domain, which determines theirsensitivity to the second messengers Ca₂ and diacylglycerol (DAG).Despite the high degree of homology, however, there is a surprisingdegree of nonredundancy. Thus, individual PKC isoforms mediate differentand unique cellular functions in different cell types and differenttissues. PKC delta belongs to the subfamily of novel isoforms (PKC δ,PKC ε, PKC θ and PKC η), which are insensitive to Ca₂. PKC delta iswidely regarded as having pro-apoptotic properties. Caspase activationmediates cleavage of PKC delta which results in release of the activecatalytic domain. In addition, PKC delta activity is known to initiate anumber of pro-apoptotic signals, such as increased expression andstability of p53 (Johnson C L, 2002 and Abbas T, 2004), mitochondrialcytochrome C release (Majumder P K, 2000 and Basu A, 2001) and c-Ablactivation. Under certain conditions however, PKC delta has beenreported to have a protective role in cell survival. PKC delta has alsobeen reported to regulate B-lymphocyte survival. PKC delta also mediatestissue fibrosis in part by stimulation the synthesis of collagen andother components of the extracellular matrix.

Knock-out experiments have shown that PKC delta-deficient mice have aderegulated immune system and develop autoimmune disease, but arefertile and grow to adulthood. Aspects of the present invention relateto the use of any or, or a combination of compounds of formula (Ia),(IIa), (IIIa), (IVa), or (V) as disclosed herein as an inhibitor of PKCdelta. In particular, a compound of (Ia), (IIa), (IIIa), (IVa), or (V)as disclosed herein selectively inhibits PKC delta over other PKCisoforms. In particular, a compound of formula (Ia), (IIa), (IIIa),(IVa), or (V) as disclosed herein as a selective PKC delta inhibitorsare advantageous over existing PKC delta inhibitors, as they are morepotent at inhibiting cell proliferation and are non-toxic to cells withnormal levels p21 Ras signaling.

Accordingly, one aspect of the present invention relates to a method fortreating a subject with a proliferative disorder, herein referred to asa cancer. In some embodiments, the method comprises determining the Rasgenotype of the tumor, that is, looking for the presence of increasedRas signaling. A subject having a tumor associated with increased Rassignaling in the tumor can be treated according to the methods asdisclosed herein, and can be administered a PKC delta inhibitor compoundof formula (Ia), (IIa), (IIIa), (IVa), or (V) as disclosed herein.

Other aspects of the present invention relate to methods for directingtreatment of a subject with a tumor. The status of the level of Rassignaling of the subject's tumor indicates a subject amenable totreatment according to the methods and composition as disclosed herein.In some embodiments, a subject is amenable to treatment according to themethods and compositions using a compound of formula (Ia), (IIa),(IIIa), (IVa), or (V) as disclosed herein where the Ras signaling of thesubject's tumor is increased relative to comparable cells.

One aspect of the present invention relates to a method for treating asubject with, or at risk for, developing a tumor which has aberrantlyincreased Ras signaling, comprising obtaining a biological sample fromthe subject; determining whether the biological sample contains cellswhich have aberrantly increased Ras signaling; and administering to thesubject a composition comprising a PKC delta inhibitor of a compound offormula (Ia), (IIa), (IIIa), (IVa), or (V) as disclosed herein to thesubject upon determination of the aberrantly increased Ras signaling, tothereby inhibit PKC delta in the cell. In one embodiment, the aberrantlyincreased Ras signaling results from one or more occurrences, includingexpression of activated Ras, over-expression of wild-type Ras, orover-activation of wild-type Ras.

Expression of activated Ras may be detected by ELISA, western blot,antibody staining, immunohistochemistry, immunofluorescence, or anycombination thereof. Alternatively, it may be detected by determinationof the presence of a mutation in a Ras nucleic acid sequence, bypolymerase chain reaction, primer-extension, allele-specific probehybridization, allele-specific primer extension, allele-specificamplification, nucleotide sequencing, 5′ nuclease digestion, molecularbeacon assay, oligonucleotide ligation assay, single-strandedconformation polymorphism, or combinations thereof.

Accordingly, in certain embodiments, the inventive compounds of formula(Ia), (IIa), (IIIa), (IVa), or (V) as disclosed herein are useful in amethod for the treatment of a proliferative disease or disorder, such ascancer or tumor. In some embodiments, the cancer is for example, abronchopulmonary cancer, a gastrointestinal cancer or a pancreaticneuroendocrine cancer, a carcinoid and/or neuroendocrine cancer, amalignant melanoma, pancreatic, gastrointestinal or lung cancer. In someembodiments, a neuroendocrine cancer can be derived from abronchopulmonary, or foregut or hindgut tumor (e.g., neuroendocrinetumor cell line of pulmonary and gastrointestinal origin).

In certain embodiments, the method involves the administration of atherapeutically effective amount of the compound or a pharmaceuticallyacceptable derivative thereof to a subject (including, but not limitedto a human or animal) in need of treatment. In some embodiments, thesubject in need of treatment has cancer, or is likely to get cancer.

In certain embodiments, the inventive compounds as useful for thetreatment of cancer, including, but not limited to, bronchopulmonarycancer, a gastrointestinal cancer or a pancreatic neuroendocrine cancer,or a carcinoid and/or neuroendocrine cancer, a malignant melanoma,pancreatic, gastrointestinal or lung cancer. In some embodiments, aneuroendocrine cancer can be derived from a bronchopulmonary, or foregutor hindgut tumor.

In some embodiments, the methods as disclosed herein compriseadministering to a subject with cancer a composition comprising acompound of formula (Ia), (IIa), (IIIa), (IVa), or (V) as disclosedherein, wherein a subject has a cancer selected from any or acombination of: glioblastoma, retinoblastoma, breast cancer, cervicalcancer, colon and rectal cancer, leukemia, lymphoma, lung cancer(including, but not limited to small cell lung cancer), melanoma and/orskin cancer, multiple myeloma, non-Hodgkin's lymphoma, ovarian cancer,pancreatic cancer, prostate cancer and gastric cancer, bladder cancer,uterine cancer, kidney cancer, testicular cancer, stomach cancer, braincancer, liver cancer, or esophageal cancer). In some embodiments, thecancer is not melanoma.

In some embodiments, the methods as disclosed herein compriseadministering to a subject with cancer a composition comprising acompound of formula (Ia), (IIa), (IIIa), (IVa), or (V) as disclosedherein, wherein a subject has a cancer selected from any or acombination of, but not limited to: breast cancer, cervical cancer,colon and rectal cancer, leukemia, lung cancer, melanoma, multiplemyeloma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer,prostate cancer, and gastric cancer, to name a few. In certainembodiments, the inventive anticancer agents are active against leukemiacells and melanoma cells, and thus are useful for the treatment ofleukemias (e.g., myeloid, lymphocytic, myelocytic and lymphoblasticleukemias) and malignant melanomas. In still other embodiments, theinventive anticancer agents are active against solid tumors.

In some embodiments, compounds of the invention are useful in thetreatment of proliferative diseases e.g., cancer, benign neoplasms,inflammatory disease, autoimmune diseases. In other embodiments, theinventive compounds are useful in the treatment of autoimmune diseases;allergic and inflammatory diseases; diseases of the central nervoussystem (CNS), such as neurodegenerative diseases (e.g. Huntington'sdisease); vascular diseases, such as restenosis; musculoskeletaldiseases; cardiovascular diseases, such as stroke; pulmonary diseases;gastric diseases; infectious diseases, and fibrotic diseases, such asChronic Kidney Disease, Liver Fibrosis, Pulmonary/Lung Fibrosis,Systemic Sclerosis, Idiopathic pulmonary fibrosis; Cystic fibrosis,Cirrhosis, Endomyocardial fibrosis (heart); Mediastinal fibrosis (softtissue of the mediastinum), Myelofibrosis (bone marrow), Retroperitonealfibrosis (soft tissue of the retroperitoneum), Progressive massivefibrosis (lungs); a complication of coal workers' pneumoconiosis,Nephrogenic systemic fibrosis (skin), Crohn's Disease (intestine),Keloid (skin), Old myocardial infarction (heart), Scleroderma/systemicsclerosis (skin, lungs), post-radiation fibrosis, and drug-inducedfibrosis.

In another aspect of the invention, methods for the treatment of acancer in a subject are provided, the method comprising administering atherapeutically effective amount of an inventive compound, as describedherein, to a subject in need thereof. In certain embodiments, a methodfor the treatment of cancer is provided comprising administering atherapeutically effective amount of an inventive compound, or apharmaceutical composition comprising an inventive compound to a subjectin need thereof, in such amounts and for such time as is necessary toachieve the desired result.

In some embodiments, U.S. Pat. Nos. 4,313,872 and 7,276.567 andInternational patent applications W02006/060196, W02005/065666,W020071106424 disclose use of PKC delta inhibitors for treatment ofcancer. However, these applications do not teach, suggest or discussusing a compound of formula (Ia), (IIa), (IIIa), or (IVa) as disclosedherein for the treatment of cancer.

In certain embodiments, the inventive compound is administeredparenterally. In certain embodiments, the inventive compound isadministered intravenously. In certain embodiments, the inventivecompound is administered topically. In certain embodiments of thepresent invention, a “therapeutically effective amount” of the inventivecompound or pharmaceutical composition is that amount effective forkilling or inhibiting the growth of tumor cells. The compounds andcompositions, according to the method of the present invention, may beadministered using any amount and any route of administration effectivefor killing or inhibiting the growth of tumor cells. Thus, theexpression “amount effective to kill or inhibit the growth of tumorcells,” as used herein, refers to a sufficient amount of agent to killor inhibit the growth of tumor cells. The exact amount required willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the infection, the particularanticancer agent, its mode of administration, and the like.

In some embodiments, the compounds as disclosed herein are useful formethods for the treatment or prevention of disorders where inhibition ofPKC delta is desirable, for example, diseases associated with impairedinsulin sensitivity or fatty liver disease (FLD), including hepaticsteatosis and type 2 diabetes, and non-alcoholic steatohepatitis (NASH),according to the methods as disclosed in International ApplicationW0/2011/041385 which is incorporated herein in its entirety byreference.

As disclosed herein the inventors have demonstrated the compounds offormula (Ia), (IIa), (IIIa), (IVa), or (V) as disclosed herein inhibitcell proliferation, in some embodiments the inventive compounds asdisclosed herein can be used in a method to prevent restenosis of bloodvessels in a subject with a trauma such as angioplasty and stenting. Forexample, encompassed herein in the invention, the compounds of formula(Ia), (IIa), (IIIa), (IVa), or (V) are useful as a coating for implantedmedical devices, such as tubings, shunts, catheters, artificialimplants, pins, electrical implants such as pacemakers, and especiallyfor arterial or venous stents, including balloon expandable stents. Incertain embodiments inventive compounds are encompassed for use incoating an implantable medical device, or alternatively, be passivelyadsorbed to the surface of the implantable device. In certain otherembodiments, the inventive compounds may be formulated to be containedwithin, or, adapted to release by a surgical or medical device orimplant, such as, for example, stents, sutures, indwelling catheters,prosthesis, and the like.

For example, drugs having antiproliferative and anti-inflammatoryactivities have been evaluated as stent coatings, and have shown promisein preventing restenosis (See, for example, Presbitero et al., “Drugeluting stents do they make the difference?”, Minerva Cardioangiol.,2002, 50(5):431-442; Ruygrok et al., “Rapamycin in cardiovascularmedicine”, Intern. Med. J., 2003, 33(3):103-109; and Marx et al., “Benchto bedside: the development of rapamycin and its application to stentrestenosis”, Circulation, 2001, 104(8):852-855, each of these referencesis incorporated herein by reference in its entirety).

Accordingly, without wishing to be bound to theory, the compounds offormula (Ia), (IIa), (IIIa), (IVa), or (V) which inhibit cellproliferation and have antiproliferative effects can be used as stentcoatings and/or in stent drug delivery devices, inter alia for theprevention of restenosis or reduction of restenosis rate in a subject.Suitable coatings and the general preparation of coated implantabledevices are described in U.S. Pat. Nos. 6,099,562; 5,886,026; and5,304,121; each of which is incorporated herein by reference. A varietyof compositions and methods related to stem coating and/or local stentdrug delivery for preventing restenosis are known in the art (see, forexample, U.S. Pat. Nos. 6,517,889; 6,273,913; 6,258,121; 6,251,136;6,248,127; 6,231,600; 6,203,551; 6,153,252; 6,071,305; 5,891,507;5,837,313 and published U.S. patent application No.: US200110027340,each of which is incorporated herein by reference in its entirety).

In some embodiments, the compounds as disclosed herein can be used in amethod to eliminate a biliary, gastrointestinal, esophageal,tracheal/bronchial, urethral, and/or vascular obstruction using a stentcoated with a composition as disclosed herein. Methods for eliminatingbiliary, gastrointestinal, esophageal, tracheal/bronchial, urethraland/or vascular obstructions using stents are known in the art. Theskilled practitioner will know how to adapt these methods in practicingthe present invention. For example, guidance can be found in U.S. PatentApplication Publication No. 2003/0004209 in paragraphs [0146]-[0155],which paragraphs are hereby incorporated herein by reference.

Additionally, the present invention provides pharmaceutically acceptablederivatives of the inventive compounds, and methods of treating asubject using such compounds, pharmaceutical compositions thereof, oreither of these in combination with one or more additional therapeuticagents.

Another aspect of the invention relates to a method of treating orlessening the severity of a disease or condition associated with aproliferation disorder in a patient, said method comprising a step ofadministering to said patient, a compound of formula (Ia), (IIa),(IIIa), (IVa), or (V) or a composition comprising said compound.

The compounds of the invention are preferably formulated in dosage unitform for ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof therapeutic agent appropriate for the patient to be treated. It willbe understood, however, that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular patient ororganism will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, sex and diet of the patient; the time ofadministration, mute of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts (see, for example, Goodmanand Gilman's The Pharmacological Basis of Therapeutics, Tenth Edition,A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Bill Press, 155-173,2001, which is incorporated herein by reference in its entirety).

Another aspect of the invention relates to a method for inhibiting PKCdelta activity in a biological sample or a patient, which methodcomprises administering to the patient, or contacting said biologicalsample with an inventive compound or a composition comprising saidcompound.

In some embodiments, a subject is selected to be administered acomposition comprising a compound of formula (Ia), (IIa), (IIIa), (IVa),or (V) where the subject is selected if they have an elevated orincreased level of Ras signaling in a biological sample obtained fromthe subject, where the biological sample is a cancer sample, or biopsysample obtained from the subject. In some embodiments, an increasedlevel of ras signaling is a statistically significant increase level ofRas signaling as compared to a normal sample (e.g., a normal tissuessample from a normal subject and/or a non-malignant or noncanceroustissue sample). Ras signaling can be measured by any means commonlyknown in the art, for example, by any means as disclosed inInternational Application W0/20071106424, which is incorporated hereinin its entirety by reference.

Activated Ras is typically detected directly (i.e., the antibody to theantigen of interest is labeled) or indirectly (i.e., a secondaryantibody that recognizes the antibody to the antigen of interest islabeled) using a detectable label. The particular label or detectablegroup used in the assay is usually not critical, as long as it does notsignificantly interfere with the specific binding of the antibodies usedin the assay. The amount of activated Ras protein in a sample can bemeasured indirectly by measuring the amount of added (exogenous)activated Ras protein displaced from a capture agent, i.e. ananti-activated Ras antibody, by the activated Ras in the sample. Innoncompetitive assays, the amount of activated Ras in a sample isdirectly measured. In some embodiments, a noncompetitive “sandwich”assay can be used to measure activated Ras where the capture agent(e.g., a first antibody) is bound directly to a solid support (e.g.,membrane, microtiter plate, test tube, dipstick, glass or plastic bead)where it is immobilized. The immobilized agent then captures any antigenof interest present in the sample. The immobilized antigen of interestcan then be detected using a second labeled antibody to the antigen ofinterest. Alternatively, the second antibody can be detected using alabeled secondary antibody that recognizes the second antibody.

In some embodiments, a method measuring the expression of activated Ras,is by antibody staining with an antibody that binds specifically to theantigen employing a labeling strategy that makes use of luminescence orfluorescence. Such staining may be carried out on fixed tissue or cellsthat are ultimately viewed and analyzed under a microscope. Stainingcarried out in this manner can be scored visually or by using opticaldensity measurements. Staining may also be carried out using either liveor fixed whole cells in solution, e.g. cells isolated from blood ortumor biopsy. Such cells can be analyzed using a fluorescence activatedcell sorter (FACS), which can determine both the number of cells stainedand the intensity of the luminescence or fluorescence. Such techniquesare well known in the art, and exemplary techniques are described inLuwor et al. ((2001), Cancer Res. 61:5355-61). One of skill in the artwill realize that other techniques of detecting expression might be moreor less sensitive than these techniques. As meant herein, cells expresslittle or no antigen if little or no antigen can be detected using anantibody staining technique that relies on luminescence or fluorescence.

Alternatively, expression of activated Ras in cells, particularly tumorcells, can be detected in vivo in a subject by introducing into thesubject a labeled antibody to activated Ras protein. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In some embodiments, immunohistochemistry (“IHC”) andimmunocytochemistry (“ICC”) techniques, for example, may be used. IHC isthe application of immunochemistry to tissue sections, whereas ICC isthe application of immunochemistry to cells or tissue imprints afterthey have undergone specific cytological preparations such as, forexample, liquid-based preparations. Immunochemistry is a family oftechniques based on the use of a specific antibody, wherein antibodiesare used to specifically target molecules inside or on the surface ofcells. The antibody typically contains a marker that will undergo abiochemical reaction, and thereby experience a change color, uponencountering the targeted molecules.

In some instances, signal amplification may be integrated into theparticular protocol, wherein a secondary antibody, that includes themarker stain, follows the application of a primary specific antibody.Immunohistochemical assays are known to those of skill in the art (e.g.,see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, etal., J. Cell. Biol. 105:3087-3096 (1987).

In some embodiments, for immunohistochemistry, tissue sections areobtained from a patient and fixed by a suitable fixing agent such asalcohol, acetone, and paraformaldehyde, to which is reacted an antibody.Conventional methods for immunohistochemistry are described in Harlowand Lane (eds) (1988) In “Antibodies A Laboratory Manual”, Cold SpringHarbor Press, Cold Spring Harbor, N.Y.; Ausbel et al. (eds) (1987), inCurrent Protocols In Molecular Biology, John Wiley and Sons (New York,N.Y.). Biological samples appropriate for such detection assays include,but are not limited to, cells, tissue biopsy, whole blood, plasma,serum, sputum, cerebrospinal fluid, breast aspirates, pleural fluid,urine and the like. For direct labeling techniques, a labeled antibodyis utilized. For indirect labeling techniques, the sample is furtherreacted with a labeled substance. Alternatively, immunocytochemistry maybe utilized. In general, cells are obtained from a patient and fixed bya suitable fixing agent such as alcohol, acetone, and paraformaldehyde,to which is reacted an antibody. Methods of immunocytological stainingof human samples is known to those of skill in the art and described,for example, in Brauer et al., 2001 (FASEB J, 15, 2689-2701),Smith-Swintosky et al., 1997. Immunological methods of the presentinvention are advantageous because they require only small quantities ofbiological material, e.g., a biopsy cancer tissue sample. Such methodsmay be done at the cellular level and thereby necessitate a minimum ofone cell. Preferably, several cells are obtained from a patient affectedwith or at risk for developing cancer and assayed according to themethods of the present invention.

In some embodiments, activated Ras can be detected by a mutationdetection kit and/or systems, including but not limited to, packagedprobe and primer sets (e.g., TaqMan probe/primer sets),arrays/microarrays of nucleic acid molecules, and beads that contain oneor more probes, primers, or other detection reagents for detectingactivated Ras mutations. The kits/systems can optionally include variouselectronic hardware components; for example, arrays (“DNA chips”) andmicrofluidic systems (“lab-on-a15 chip” systems, biomedicalmicro-electro-mechanical systems (bioMEMs), or multicomponent integratedsystems) provided by various manufacturers typically comprise hardwarecomponents. Other kits/systems (e.g., probe/primer sets) may not includeelectronic hardware components, but may be comprised of, for example,one or more mutation detection reagents (along with, optionally, otherbiochemical reagents) packaged in one or more containers.

In some embodiments, the subject administered a composition comprising acompound of formula (Ia), (IIa), (IIIa), (IVa), or (V) has aberrantlyincreased Ras signaling, e.g., as a result of expression of activatedRas. In some embodiments, aberrantly increased Ras signaling isdetermined by detection of an activated Ras protein or a nucleotidesequence encoding an activated form of Ras protein.

In some embodiments, increased Ras signaling is associated withactivation of a pathway selected from the group consisting of Raf1/MAPK,RasGDS/Ras/Rho, PI3K, and combinations thereof. In some embodiments,activated form of Ras is selected from the group consisting of K-ras,H-ras, and N-ras. In some embodiments, aberrantly increased Rassignaling results from over-expression of wild-type Ras, or fromincreased activation of one or more effector pathways downstream of Ras.

Furthermore, after formulation with an appropriate pharmaceuticallyacceptable excipient in a desired dosage, the pharmaceuticalcompositions of this invention can be administered to humans and otheranimals orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,creams or drops), bucally, as an oral or nasal spray, or the like,depending on the severity of the infection being treated. In certainembodiments, the compounds of the invention may be administered atdosage levels of about 0.001 mg/kg to about 50 mg/kg, from about 0.01mg/kg to about 25 mg/kg, or from about 0.1 mg/kg to about 10 mg/kg ofsubject body weight per day, one or more times a day, to obtain thedesired therapeutic effect. It will also be appreciated that dosagessmaller than 0.001 mg/kg or greater than 50 mg/kg (for example 50-100mg/kg) can be administered to a subject. In certain embodiments,compounds are administered orally or parenterally.

Uses

The present invention provides novel compounds useful in the treatmentof diseases or disorders associated with PKC delta activity. Thecompounds are useful in the treatment of diseases or condition thatbenefit from inhibition of PKC delta. In certain embodiments, theinventive compounds as useful for the treatment of cancer, including,but not limited to, bronchopulmonary cancer, a gastrointestinal canceror a pancreatic neuroendocrine cancer, or a carcinoid and/orneuroendocrine cancer, a malignant melanoma, pancreatic,gastrointestinal or lung cancer. In some embodiments, a neuroendocrinecancer can be derived from a bronchopulmonary, or foregut or hindguttumor.

In certain embodiments, the inventive compounds are useful in thetreatment of cellular proliferative diseases, such as cancer (e.g.,cutaneous T-cell lymphoma) or benign proliferative diseases such asfibrotic diseases including systemic sclerosis, radiation inducedfibrosis and pulmonary fibrosis; autoimmune diseases; allergic andinflammatory diseases; diseases of the central nervous system (CNS),such as neurodegenerative diseases (e.g. Huntington's disease); vasculardiseases, such as restenosis; musculoskeletal diseases; cardiovasculardiseases; stroke; pulmonary diseases; gastric diseases; and infectiousdiseases.

In certain embodiments, the compounds of the present invention areuseful as inhibitors of PKC delta and thus are useful asantiproliferative agents, and thus may be useful in the treatment ofcancer, by effecting tumor cell death or inhibiting the growth of tumorcells, and in other diseases characterized by excessive proliferation ofcells, such as fibrotic diseases, by inhibiting the proliferation ofcells contributing to fibrosis or collagen production by these cells. Incertain exemplary embodiments, the inventive compounds are useful in thetreatment of cancers and other proliferative disorders, including, butnot limited to breast cancer, cervical cancer, colon and rectal cancer,leukemia, lung cancer, melanoma, multiple myeloma, non-Hodgkin'slymphoma, ovarian cancer, pancreatic cancer, prostate cancer, andgastric cancer, to name a few. In certain embodiments, the inventiveanticancer agents are active against leukemia cells and melanoma cells,and thus are useful for the treatment of leukemias (e.g., myeloid,lymphocytic, myelocytic and lymphoblastic leukemias) and malignantmelanomas. In certain embodiments, the inventive compounds are activeagainst cutaneous T-cell lymphoma. Additionally, as described herein,the inventive compounds may also be useful in the treatment of protozoalinfections. Additionally, as described herein, the inventive compoundsmay also be useful in the treatment of autoimmune or inflammatorydiseases and fibrotic diseases. Furthermore, as described herein, theinventive compounds may also be useful in the treatment ofneurodegenerative diseases. As described herein, the inventive compoundsmay also be useful in the treatment of cardiovascular diseases.

Activated Ras proteins play a key role in the development of many humancancers. “Ras” and “p21Ras” are used interchangeably herein. Mutationsin Ras are observed in approximately one third of all tumors (Bos,Cancer Res 49:4682-4689 [1989]; and Clark and Der, in GTPases in Biology[eds. Dickey and Birmbauer], Springer-Verlag London Ltd., pp. 259-287[1993]). Indeed, the frequency of Ras mutation approaches 100% in sometypes of tumors (e.g., pancreatic adenocarcinoma). These mutated Rasproteins demonstrate decreased inherent GTPase activity, and areresistant to the action of GTPase-activating proteins (GAPs). Thus,these mutations, e.g., mutations localized in codons 12, 13, 59, 61, 63,116, 117, and 146, are activating mutations resulting in the Ras proteinbeing locked in an active conformation, leading ultimately toinappropriate cell proliferation signaling. Furthermore, activated formsof the Ras protein are useful in the induction of tumors, therebyproviding direct evidence for Ras involvement in malignant celltransformation and tumorigenesis. Moreover, deletion of the activatedRas gene from tumor cell lines impairs their tumorigenicity (Paterson etal., Cell 51:803-812 [1987]; and Shirasawa et al., Science 260:85-88[1993]). Three closely related Ras genes are H-ras (GenBank AccessionNo. NM_005343, K-ras (GenBank Accession No. NM_004985;) and N-ras(GenBank Accession No. NM_002524). Wild-type Ras proteins, found innormal, healthy individuals, cycle between an active (GTP bound) stateand an inactive (GDP bound) state. Activated Ras proteins result fromactivating mutations which have decreased inherent GTPase activity, andare resistant to the action of GTPase-activating proteins (GAPs), thenatural negative regulators of Ras proteins. Thus, these mutations,e.g., mutations localized in codons 12, 13, 59, 61, 63, 116, 117, and146, are activating mutations resulting in the Ras protein being lockedin an active conformation. The presence of the activated form of Ras ina cell leads ultimately to inappropriate cell proliferation signaling.

Activated Ras proteins play a key role in the development of many humancancers. Such mutations in Ras are observed in approximately one thirdof all tumors (Bos, Cancer Res 49:4682-4689 [1989]; and Clark and Der,in GTPases in Biology [eds. Dickey and Birmbauer], Springer-VerlagLondon Ltd., pp. 259-287 [1993]). Indeed, the frequency of Ras mutationapproaches 100% in some types of tumors (e.g., pancreaticadenocarcinoma).

In addition to the correlation between the presences of activated Rasmutations in a high percentage of a variety of cancers, a wealth ofexperimental evidence indicates that increased Ras activity is involvedin malignant cell transformation and tumorigenesis. For example,activated forms of the Ras protein can be used to experimentallytransform cells in culture and induce tumors in animal models.Furthermore, deletion of the activated Ras gene from tumor cell linesimpairs their tumorigenicity (Paterson et al., Cell 51:803-812 [1987];and Shirasawa et al., Science 260:85-88 [1993]).

In certain exemplary embodiments, the compounds of the invention areuseful for treatment of diseases and disorders where there is aberrantor increased Ras signaling. Such disorders are disclosed inInternational Application W02007/0106424, and US patent applicationUS2009/0330503, both incorporated herein in their entirety by reference.Aberrant signaling through Ras signaling pathways occurs as a result ofseveral different classes of mutational damage in tumor cells. Table 1provides a list of methods of increased activation of Ras signalingpathway in different tumors, and encompasses cancers amenable to betreated according to the methods and compositions as disclosed herein.

TABLE 1 Activation of RAS signaling pathways in different tumors Defector Mutation Tumor Type Frequency RAS mutation Pancreas 90 (K) Lungadenocarcinoma 35 (K) (non-small-cell) Colorectal 45 (K) Thyroid(follicle) 55 (H, K, N) Thyroid 60 (H, K, N) (undifferentiated,papillary) Seminoma 45 (K, N) Melanoma 34 (N) Bladder 10 (H) Liver 30(N) Kidney 10 (H) Myelodysplastic syndrome 40 (N, K) Acute myelogenousleukemia 30 (N) BRAF mutation Melanoma 66 Colorectal 12 EGFRoverexpression Most carcinomas >50 ERBB2 amplification Breast 30 PTENloss Glioblastoma multiforme 20-30 Prostate 20 Pancreas 40 AKT2amplification Ovarian 12 Pancreas 10 PI3K amplification Ovarian 40 EGFR= epidermal growth factor receptor PI3K = phosphotidylinositol-3-kinaseH, K and N refer to HRAS, KRAS and NRAS respectively Table from:Downward J. Targeting RAS signaling pathways in cancer therapy. Nat.Rev. Cancer. 2003 Jan.; 3(1): 11-22.

As detailed in the exemplification herein, in assays to determine theability of compounds to inhibit PKC delta activity, certain inventivecompounds may exhibit IC50 values <100 μM. In certain other embodiments,inventive compounds exhibit IC50 values <50 μM. In certain otherembodiments, inventive compounds exhibit IC50 values <40 μM. In certainother embodiments, inventive compounds exhibit IC50 values <<30 μM. Incertain other embodiments, inventive compounds exhibit IC50 values <20μM. In certain other embodiments, inventive compounds exhibit IC50values <10 μM. In certain other embodiments, inventive compounds exhibitIC50 values <7.5 μM. In certain other embodiments, inventive compoundsexhibit IC50 values <5 μM. In certain other embodiments, inventivecompounds exhibit IC50 values <2.5 μM. In certain other embodiments,inventive compounds exhibit IC50 values <1 μM. In certain otherembodiments, inventive compounds exhibit IC50 values <0.75 μM. Incertain embodiments, inventive compounds exhibit IC50 values <0.5 μM. Incertain other embodiments, inventive compounds exhibit IC50 values <50.25 μM. In certain embodiments, inventive compounds exhibit IC50 values<0.1 μM. In certain embodiments, inventive compounds exhibit IC50 values<75 nM. In certain embodiments, inventive compounds exhibit IC50 values<50 nM. In certain embodiments, inventive compounds exhibit IC50 values<25 nM. In certain embodiments, inventive compounds exhibit IC50 values<10 nM.

In assays to determine the ability of compounds to inhibit cancer cellgrowth certain inventive compounds may exhibit IC50 values <100 μM. Incertain other embodiments, inventive compounds exhibit IC50 values <50μM. In certain other embodiments, inventive compounds exhibit IC50values <40 μM. In certain other embodiments, inventive compounds exhibitIC50 values <30 μM. In certain other embodiments, inventive compoundsexhibit IC50 values <20 μM. In certain other embodiments, inventivecompounds exhibit IC50 values <10 μM. In certain other embodiments,inventive compounds exhibit IC50 values <7.5 μM. In certain embodiments,inventive compounds exhibit IC50 values <5 μM. In certain otherembodiments, inventive compounds exhibit IC50 values <2.5 μM. In certainembodiments, inventive compounds exhibit IC50 values <1 μM. In certainembodiments, inventive compounds exhibit IC50 values <0.75 μM. Incertain embodiments, inventive compounds exhibit IC50 values <0.5 μM. Incertain embodiments, inventive compounds exhibit IC50 values <0.25 μM.In certain embodiments, inventive compounds exhibit IC50 values <0.1 μM.In certain other embodiments, inventive compounds exhibit IC50 values<75 nM. In certain other embodiments, inventive compounds exhibit IC50values <50 nM. In certain other embodiments, inventive compounds exhibitIC50 values <25 nM. In certain other embodiments, inventive compoundsexhibit IC50 values <10 nM. In other embodiments, exemplary compoundsexhibited IC50 values <7.5 nM. In other embodiments, exemplary compoundsexhibited IC50 values <5 nM.

In connection with the administration of the drug, an “effective amount”indicates an amount that results in a beneficial effect for at least astatistically significant fraction of patients, such as an improvementof symptoms, a cure, a reduction in disease load, reduction in tumormass or cell numbers, extension of life, improvement in quality of life,or other effect generally recognized as positive by medical doctorsfamiliar with treating the particular type of disease or condition.

The effective dosage of active ingredient employed may vary depending onthe particular compound employed, the mode of administration and theseverity of the condition being treated. The skilled artisan is aware ofthe effective dose for each patient, which may vary with diseaseseverity, individual genetic variation, or metabolic rate. However, ingeneral, satisfactory results are obtained when the compounds of theinvention are administered at a daily dosage of from about 0.5 to about1000 mg/kg of body weight, optionally given in divided doses two to fourtimes a day, or in sustained release form. The total daily dosage isprojected to be from about 1 to 1000 mg, preferably from about 2 to 500mg. Dosage forms suitable for internal use comprise from about 0.5 to1000 mg of the active compound in intimate admixture with a solid orliquid pharmaceutically acceptable carrier. This dosage regimen may beadjusted to provide the optimal therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. The route of administration may be intravenous (I.V.),intramuscular (I.M.), subcutaneous (S.C.), intradermal (ID.),intraperitoneal (I.P.), intrathecal (I.T.), intrapleural, intrauterine,rectal, vaginal, topical, intratumor and the like. The compounds of theinvention can be administered parenterally by injection or by gradualinfusion over time and can be delivered by peristaltic means.

Administration may be by transmucosal or transdermal means. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives. Inaddition, detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays, for example, or usingsuppositories. For oral administration, the compounds of the inventionare formulated into conventional oral administration forms such ascapsules, tablets and tonics. For topical administration, thepharmaceutical composition (inhibitor of kinase activity) is formulatedinto ointments, salves, gels, or creams, as is generally known in theart. The therapeutic compositions of this invention, e.g., PKC deltainhibitors, are conventionally administered intravenously, as byinjection of a unit dose, for example.

The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluents; i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual. Therapeuticcompositions useful for practicing the methods of the present invention,e.g. PKC delta inhibitors, are described herein. Any formulation or drugdelivery system containing the active ingredients, which is suitable forthe intended use, as are generally known to those of skill in the art,can be used. Suitable pharmaceutically acceptable carriers for oral,rectal, topical or parenteral (including inhaled, subcutaneous,intraperitoneal, intramuscular and intravenous) administration are knownto those of skill in the art.

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention.

EXAMPLES

Cell Lines:

BON1, a human foregut (pancreatic) carcinoid tumor cell line (Parekh etal. 1994) was obtained from Kjell Oberg (Uppsala University, Sweden)through Dr. Evan Vosburgh. H727 cells, derived from a humanbronchopulmonary carcinoid tumor (Schuller et al. 1987), were purchasedfrom ATCC. The CNDT 2.5 cell line, initially described as a human midgutcarcinoid tumor cell line (Van Buren et al. 2007), was provided by Dr.Lee Ellis, (MD Anderson Cancer Center). The provenance of this cell lineis currently under review by the originator. BONI, H727, MCF10 and CNDT2.5 cells were propagated in 10% FBS (Invitrogen); Dulbecco'sModification of Earle's Media/Hams F-12 50:50 media (Cellgro); 2 mML-Glutamine (Invitrogen); 200 U Penicillin/ml; 200 μg Streptomycin/ml(Invitrogen); 10 ng/ml Nerve Growth Factor (Invitrogen); 1×MEMNon-Essential Amino Acids (Cellgro); 1×MEM Vitamin Solution (Cellgro); 1mM Sodium Pyruvate; 0.015 M HEPES buffer (pH7.3) (AmericanBioanalytical).

Clonogenic Assays:

100,000 cells were seeded on 100 mm dishes 5 with 10 ml media per dish(Li et al. 2004). On day 4, cells were treated with a PKC deltainhibitor, or vehicle control for 6, 18, 24 or 48 hours. Cells weretrypsinized; counted via Trypan Blue Exclusion Method in order todetermine the number of live cells in the sample, and 500 live cellswere seeded in triplicate onto 6 well plates. Cells were monitored forappropriate colony size and re-fed every three to four days. At Day 17,cells were stained with ethidium bromide (Guda et al. 2007) and countedusing UVP LabWorks software.

PKC Kinase Activity Assays:

Assays were carried out using recombinant PKC alpha or PKC gamma,(Invitrogen) and the Omnia® Kinase Assays (Invitrogen) with a “PKCkinase-specific” peptide substrate. Incorporation of achelation-enhanced fluorophore (CHEF) results in an increase influorescence ex360/em485) upon phosphorylation. The kit was usedaccording to the manufacturer's instructions.

Reagents:

Rottlerin was purchased from (EMD Biosciences). The PKC delta inhibitorKAMI is a chimeric molecule combining the chromene portion of rottlerinwith the carbazole portion of staurosporine.

Cell Proliferation Assays—

Cell proliferation was assessed using an MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay(Roche, Mannheim, Germany). The number of viable cells growing in asingle well on a 96-well microtiter plate was estimated by adding 10 μlof MTT solution (5 mg/ml in phosphate-buffered saline [PBS]). After 4 hof incubation at 37° C., the stain is diluted with 100 μl of dimethylsulfoxide. The optical densities are quantified at a test wavelength of570 nm and a reference wavelength of 690 nm on a multiwellspectrophotometer. In some assays, MTS was used as substrate (Promega,Madison, Wis.), and the absorbance of the product was monitored at 490nm.

Cytotoxicity Assay:

LDH release was assessed by spectrophotometrically measuring theoxidation of NADH in both the cells and media. Cells were seeded in24-well plates, and exposed to PKC delta inhibitors or vehicle. Afterdifferent times of exposure, cytotoxicity was quantified by a standardmeasurement of LDH release with the use of the LDH assay kit (RocheMolecular Biochemicals) according to the manufacturer's protocol.Briefly, total culture medium was cleared by centrifugation. For assayof released LDH, supernatants were collected. To assess total LDH incells, Triton X-100 was added to vehicle (control) wells to releaseintracellular LDH. LDH assay reagent was added to lysates orsupernatants and incubated for up to 30 min at room temperature in dark,the reaction was stopped, and the absorbance was measured at 490 nm. Thepercentage of LDH release was then calculated as the LDH in thesupernatants as a fraction of the total LDH. Immunoblot Analyses:

Levels of proteins were measured and quantitated in carcinoid celllines, as we have previously reported (Xia et al. 2007). Harvested cellswere disrupted in a buffer containing 20 mM Tris (pH 7.4), 0.5% NP-40,and 250 mM NaCl. Total protein (40 μg) was separated on 10%SDS-polyacrylamide gels and transferred to nitrocellulose membranes orPVDF membranes. Membranes are blocked overnight and probed withaffinity-purified antibodies against PKC alpha and delta (BDTransduction Lab), or betta actin (Sigma). After washing, the blots wereincubated with horseradish peroxidase conjugated secondary antibodiesand visualized using the Amersham enhanced chemiluminescence ECL system,and quantitated by digital densitometry. Antibodies against human ERK,phospho-ERK, AKT and phospho-Ser473-AKT were purchased from CellSignaling (Danvers, Mass.). GTP-bound Ras was assayed by affinitypurification using a Raf-1/RBD agarose conjugate (Upstate Biotechnology,Lake Placid, N.Y.), and detected with a pan-Ras antibody (CellSignaling, Danvers, Mass.), following the manufacturer's instructions.

Down-Regulation of PKC Delta by siRNA and Lentiviral Vectors:

siRNA knockdown of PKC delta and PKC alpha: siRNA duplexes for PKC delta(siRNAs) are obtained from Qiagen (Valencia, Ca). The siRNA sequencesfor targeting PKC delta are PKC delta-siRNA-1(5′-GAUGAAGGAGGCGCUCAGTT-3′; SEQ ID NO 1) and PKC delta siRNA-2(5′-GGCUGAGUUCUGGCUGGA-CTT-3′; SEQ ID NO 2). The corresponding scrambledsiRNAs were used as negative control. These siRNA sequences were alsocloned into the pRNA6.1-Neo vector with a GFP tag according to themanufacturer's instructions (GenScript, Piscataway, N.J.). siRNA for PKCalpha (PKCPKC alpha-V6) are purchased from Upstate (Lake Placid, N.Y.).Transfection of siRNA (oligo) is performed using 50 nM PKC delta siRNA,or the same amount of scrambled siRNA and Lipofectamine 2000(Invitrogen, Carlsbad, Calif.), according to the manufacturer'sinstructions. Transfection of plasmid-based siRNA vectors are carriedout using the same method. PKC delta protein levels were determined byimmunoblot analysis (see below). The lentiviral vectors were previouslydescribed (Xia et al. 2009).

Statistical Analysis.

Experiments were carried out in triplicate for all experimentalconditions. Data are shown as mean±SD. Where applicable, a two-tailedStudent's t test was performed on the means of two sets of sample dataand considered significant if p<0.05.

Example 1 PKCd Depletion by siRNA Inhibits Proliferation and InducesCytotoxicity in Human Neuroendocrine Cell Lines

To determine the effects of specific PKC delta depletion on theproliferation and survival of human neuroendocrine tumor cell lines, weused PKC delta-specific siRNA to knock-down PKC delta mRNA/protein. Cellline studied for sensitivity included BONI, a human foregut (pancreatic)carcinoid tumor cell line; H727 cells, derived from a humanbronchopulmonary carcinoid tumor; and the CNDT 2.5 cell line, a humancell line with neuroendocrine markers, initially described as a humanmidgut carcinoid tumor cell line. Exposure of the BONI and CNDT celllines to PKC delta-specific siRNA in culture resulted in a profoundinhibition of proliferation (FIG. 1). In contrast, exposure of the samecells to a control (scrambled siRNA) did not affect proliferation.Efficient knockdown of PKC delta protein by specific siRNA was verifiedby immunoblotting. To confirm and extend these experiments, lentiviralvectors containing the same shRNA sequences (PKC delta-specific orscrambled) were constructed. Infection of the BON1, H727 and CNDT celllines cell lines with these vectors demonstrated PKC delta-specificinhibition of proliferation (FIG. 2A-C). The lentiviral vectorcontaining the scrambled sequence (control) consistently had a modestinhibitory effect on proliferation of both cell lines, but this neverreached statistical significance. Efficient knockdown of PKC deltaprotein by specific siRNA was verified by immunoblotting. To determineif the inhibition of tumor cell proliferation by PKC delta knockdown wasaccompanied by cytotoxic effects on the tumor cells, cytotoxicity inthese cell lines was evaluated by quantitating LDH release. Lactosedehydrogenase (LDH), a stable cytoplasmic enzyme, is rapidly releasedinto the cell culture medium after damage of the plasma membrane, andits level correlates quantitatively with the extent of cytotoxicity.Significant increases in LDH release I cytotoxicity were detected within24 hr of exposure to the lentiviral vector containing the PKC deltashRNA, and this release increased to approach the maximum possible LDHrelease (complete cell lysis, positive control) by 72 hr (FIG. 3A-C).Only modest, but detectable, increases in LDH release were induced bythe control lentiviral vector.

Example 2 Small Molecule Inhibitors of PKC Delta are Cytotoxic toNeuroendocrine Tumor Cell Lines

We next determined whether a series of small-molecule PKC deltainhibitors would inhibit the growth of human neuroendocrine tumor celllines. While not as specific for the PKC delta isozyme as technologyemploying genetic knockdown of the PKC delta mRNA and protein, suchsmall-molecule inhibitors are more relevant for eventual therapeuticapplication. Rottlerin is a naturally-occurring product which inhibitspurified PKC delta at an IC50 of 1-3 mM in vitro, and inhibits PKC deltain cultured cells with an IC50 of 5 μM in vivo. It is relativelyselective for PKC delta (PKC delta/PKC alpha (IC 50:IC50 is >1:30)(Gschwendt et al. 1994; Kikkawa et al. 2002), and confirmed in our invitro assays (not shown). Furthermore, this compound not only directlyinhibits purified PKC delta, but also, over longer periods of exposure,significantly down-regulates PKC delta protein specifically in cells,while having no effect on the levels of other PKC isozymes (Xia et al.2007). Exposure to rottlerin produced a dose- and time-dependentdecrease in cell number in the BON1, the CNDT 2.5, and the H727 celllines, with an IC50 of approximately 5 μM by 48 hrs (not shown), and asignificant reduction in relative cell numbers by 72 hrs at the highestconcentrations tested (FIG. 4). In contrast, we have previouslydemonstrated that exposure to rottlerin under these same cultureconditions has no significant effect on the growth of nontumorigenicmurine or human cells in culture (Xia et al. 2007). Docking studies wereconducted to predict how rottlerin binds to PKC delta. Rottlerin wasdocked into the catalytic binding site of several different PKC crystalstructures. The structure of PKC θ complexed with staurosporine (pdbcode 1XJD) was selected as the most suitable model. It is known fromcrystal structures of many kinase/inhibitor complexes that the kinaseactive site is flexible, therefore, regions known to be flexible wereallowed to be free during the docking procedures. Chimeric moleculeswere designed using the PKC delta model developed from the rottlerindocking studies. The strategy was to retain most of the chromene part ofrottlerin, which is assumed to give rottlerin its specificity but tovary the “head group” which is assumed to bind to the hinge region ofthe kinase active site. A novel PKC delta inhibitor, KAM1, which is achimeric molecule containing the substituted chromene portion ofrottlerin and the N-alkylated carbazole portion of staurosporine (anon-selective pan-PKC inhibitor) (FIG. 5), was next tested for cytotoxiceffects on neuroendocrine tumor cells. Comparative analyses of PKC deltainhibitory activity showed an IC50 of 0.2 mM for rottlerin and an IC50of 0.9 μM for KAM1. In contrast, the PKC alpha IC50 was greater than 50μM for each compound, demonstrating some specificity for the novelisozyme PKC delta over classic isozyme PKC alpha. KAM1 produced a dose-and time-dependent decrease in cell number in the BON1, the CNDT 2.5,and the H727 cell lines, with an IC50 of approximately 12 μM by 48 hrs(not shown), and an 80% reduction in cell numbers by 72 hrs at thehighest concentrations tested (FIGS. 4A & B). In parallel, cytotoxicity,as assessed by LDH release, was induced by exposure of the H727 cells torottlerin, with cytotoxicity increasing as a function of time andconcentration of this inhibitor (FIG. 6). Whether neuroendocrine tumorcell lines could escape from the anti-tumor actions of PKC inhibitorswas explored by long-term exposure to the inhibitors, in twoexperimental designs. In the first, cells were plated at a lower densityto allow monitoring over longer periods for potential growth. In these“continuous” treatment studies, a PKC delta inhibitor was added at a“suboptimal” concentration, and effects on proliferation were observedas far as 144 hr after exposure (FIGS. 7 A & B). The decrease observedin the MTS signal from the control (vehicle-treated) cells at 144 hrrepresented both overgrowth of these cultures and exhaustion of theculture media. To allow evaluation over even longer periods of exposure,other cultures were re-fed with fresh growth medium containing the samePKC delta inhibitor at the same concentration. In these studies,growth-inhibitory effects persisted to 168 hr of cumulative exposure(FIGS. 7 C & D). The length of exposure to PKC delta inhibition requiredfor anti-tumor activity was next assessed. neuroendocrine tumor lineswere exposed to a sub-optimal concentration of a PKC delta-inhibitor fordifferent intervals of time, the inhibitor was then washed out of theculture, and the effects on cell growth were assessed over the next 72hrs. Differences in proliferation between rottlerin- and vehicle-treatedcultures became statistically significant by 24 hr of exposure, andremained significant for all longer periods of exposure (FIG. 8).

LDH release assesses cytotoxic damage sufficient to compromise membraneintegrity over a relatively short time-span. An alternative method,which assesses lethal, but not necessarily immediate, cumulative damageto the tumor cell is a clonogenic assay. In this assay, tumor cellswhich remain viable after exposure to the compound are tested for theirability to proliferate sufficiently over time to form colonies of tumorcells. H727 treated with vehicle or a PKC delta inhibitor at sub-optimalconcentrations varying durations. After replating of viable cells inmedia without inhibitor, colony numbers were quantitated over time.

Significant effects of the PKC delta-inhibitors on effects on reducingclonogenic capacity reached significance after as little as 6 hr ofexposure, and remained significant for all subsequent exposure times(FIG. 9). BON1 cells showed a similar drop-off in clonogenic capacity,reaching significance between 12 and 24 hr of exposure to PKC deltainhibitors.

Example 3 Ras Signaling in Neuroendocrine Tumor Cell Lines

Because of their sensitivity to PKC delta inhibition and “Ras-mediatedapoptosis”, the activity of p21Ras in these neuroendocrine tumor celllines was assessed by affinity pull-down of GTP-bound p21Ras species.Endogenous Ras activity was high in the H727 cells, was only modestlyelevated BON1 cells, and was not evident in the CNDT line, whichcontained GTP20 bound p21Ras levels comparable to those found innon-transformed cells (FIG. 10A).

It has been previously demonstrated that aberrant activation of certainRas signaling pathways, including the PI3K-AKT pathway or the Raf-MAPKpathway, are sufficient to render tumor cells susceptible to PKC deltainhibition (Xia et al. 2007). The activation status of downstreamelements of these signaling pathways was therefore explored in theseneuroendocrine tumor cell lines. Evidence for activation of MAPK, asdefined by relative elevation of phospho-ERK levels, was observed in theH727 and CNDT lines (compared to the non-transformed negative-controlcell line MCF10) (FIG. 10B). Evidence for some activation of PI3K,relative to the non-transformed negative control cell line MCF10, asdefined by activating phosphorylation of AKT (Ser473), was observed inall three neuroendocrine tumor cell lines.

Example 4 Exemplary Synthesis

These examples are for illustration only and one of ordinary skill inthe art would know how to modify the synthetic sequence to obtain thesame or different compounds of the invention (see FIG. 12E).

Unless otherwise noted, all reagents were obtained from commercialsuppliers and were used without further purification. All air ormoisture sensitive reactions were performed under a positive pressuredof argon in flame-dried glassware. Tetrahydrofuran (THF), toluene,diethyl ether (Et₂O), dichloromethane, benzene (PhH), acetonitrile(MeCN), triethylamine (NEt₃), pyridine, diisopropyl amine, methanol(MeOH), dimethylsulfoxide (DMSO), and N,N-dimethylformamide (DMF) wereobtained from a dry solvent system (Ar) degassed solvents deliveredthrough activated alumina columns, positive pressure of argon). Columnchromatography was performed on Merck silica gel Kieselgel 60 (230-400mesh). ¹HNMR and ¹³CNMR spectra were recorded on Varian 300, or 400 MHzspectrometers. Chemical shifts are reported in ppm relative to CHCl₃ at6 7.27 (¹HNMR) and δ 77.23 (¹³CNMR). Mass spectra were obtained onFisons VG Autospec. IR spectra were obtained from thin films on a NaClplate using a Perkin-Elmer 1600 series FT-IR spectrometer. See Compound2, FIG. 12F.

Synthesis of 6-bromo-2,2-dimethyl-2H-chromene-8-carbaldehyde (2)

To a 100 mL flame dried round bottomed flask containing 5.54 mL (57.2mmol, 1.15 equiv) 2-methylbut-3-yn-2-ol dissolved in 50 mL dry MeCN at0° C. was added 11.1 mL (74.6 mmol, 1.5 equiv.) DBU followed by thedropwise addition of 8.08 mL (57.2 mmol, 1.15 equiv.) freshly distilledTFAA. The reaction was stirred at 0° C. for 30 min before being addedvia cannula to a 250 mL round bottomed flask containing 10.0 g (49.7mmol, 1 equiv.) 5-bromo-2-hydroxybenzaldehyde, 9.65 mL (64.6 mmol, 1.3equiv.) DBU, and 8.5 mg (0.050 mmol, 0.001 equiv.) CuCh-2H₂O dissolvedin dry MeCN at −5° C. The reaction was stirred for 16 hr at ambienttemperature before being concentrated under reduced pressure. Theresulting residue was taken up in EtOAc, washed once with H₂0, once with1 M HCl, and once with brine before being dried over Na₂S0₄, andconcentrated. This residue was subjected to silica gel flashchromatography eluting with 19:1 to 4:1 hex/EtOAc to yield 11.17 g (84%)of the desired product (2) as a pale yellow solid. See Compound 3, FIG.12F. ¹HNMR (300 MHz, CDCl₃) δ 10.27 (s, 1H), 7.88 (s, 1H), 7.55 (d,J=8.4, 1H), 7.38 (d, J=8.4, 1H), 2.63 (s, 1H), 1.67 (s, 6H);); ¹³CNMR(75 MHz, CDCl₂) δ 188.8, 157.4, 137.5, 130.9, 130.2, 122.8, 116.1, 84.7,76.3, 74.5, 29.7; IR (NaCl, film) 3294, 1687, 1588, 1471 cm⁻¹; HRMS(+TOF) 267.0015 calcd for C₁₂H₁₂BrO₂ [M+H]⁺. found: 267.0016. R_(f)=0.38(5 9:1 hex./EtOAc).

Synthesis of 6-bromo-2,2-dimethyl-2H-chromene-8-carbaldehyde (3)

To an 80 mL microwave reaction vessel containing 4.00 g (14.8 mmol, 1equiv.) 5-bromo-2-((2-methylbut-3-yn-2-yl)oxy)benzaldehyde dissolved in60 mL dry MeCN was added 66.0 mg (0.300 mmol, 0.02 equiv.) BHT. Thereaction was heated in a microwave reactor to 180° C. for 20 min beforebeing concentrated and purified by silica gel flash chromatographyeluting with 19:1 hex./EtOAc to yield 2.10 g (53%) of the desiredproduct (3) as a yellow oil. See Compound 4, FIG. 12F. ¹HNMR (300 MHz,CDCl₃) δ 10.35 (s, 1H), 7.73 (d, J=2.7, 1H), 7.27 (dd, J=2.7, 0.3, 1H),6.29 (d, J=9.9, 1H), 5.75 (d, J=9.9, 1H), 1.50 (s, 3H); ¹³CNMR (75 MHz,CDCl₃) δ 188.0, 155.3, 134.3, 132.8, 129.4, 125.6, 124.6, 120.8, 113.4,78.4, 28.4; IR (NaCl, film) 2863, 1678, 1574 cm⁻¹; HRMS(+TOF) 267.0015calcd for C₁₂H₁₂BrO₂ [M+H]⁺. found: 267.0012. R_(f)=0.33 (9:1hex./EtOAc).

Synthesis of1-(6-bromo-2,2-dimethyl-2H-chromen-8-yl)-3-phenylprop-2-yn-1-ol (4)

To a flame dried 100 mL round bottomed flask containing 745 mg (7.30mmol, 1.3 equiv.) ethynylbenzene dissolved in 30 mL dry THP at −78° C.was dropwise added 4.2 mL (6.7 mmol, 1.2 equiv) of a 1.6 M solution ofn-BuLi in hexanes. The reaction was allowed to stir at −78° C. for 30min before the addition of 1.50 g (5.62 mmol, 1 equiv.)6-bromo-2,2-dimethyl-2Hchromene-8-carbaldehyde dissolved in 10 mL dryTHF. After stirring for 30 min at −78° C. the reaction was poured intosaturated NH₄Cl(aq)₂, extracted into EtOAc, dried over Na₂SO₄, andconcentrated. The resulting residue was purified by silica gel flashchromatography eluting with 4:1 hex./EtOAc yielding 2.1 g (99%) of thedesired product (4) as a yellow oil. See Compound 5, FIG. 12F. ¹HNMR(300 MHz, CDCl3) δ 7.52 (d, J=2.4, 1H), 7.46 (m, 2H), 7.33 (m, 3H), 7.10(d, J=2.4, 1H), 6.27 (d, J=9.9, 1H), 5.78 (s, 1H), 5.69 (d, J=9.6, 1H),3.06 (bs, 1H), 1.49 (s, 3H), 1.48 (s, 3H); ¹³CNMR (75 MHz, CDCl₃) δ149.5, 132.2, 132.0, 130.3, 130.1, 129.1, 128.7, 128.5, 123.6, 122.7,121.4, 113.1, 88.2, 86.3, 77.8, 61.3, 28.2, 28.2; IR (NaCl, film) 3428br, 2230 cm⁻¹; HRMS (+TOF) 351.0379 calcd. for C₂₀H₁₆BrO [M−H₂O]⁺.found: 351.0389. R_(f)=0.40 (4:1 hex./EtOAc).

Synthesis of (E)-1-(6-bromo-2,2-dimethyl-2H-chromen-8-yl)-3-phenylprop-52-en-1-ol (5)

To a 10 mL round bottomed flask containing 100 mg (0.271 mmol, 1 equiv.)1-(6-bromo-2,2-dimethyl-2H-chromen-8-yl)-3-phenylprop-2-yn-1-oldissolved in 1.5 mL dry THP was added 12 mg (0.33 mmol, 1.2 equiv.)LiAlH₄. The reaction was heated to reflux for 1 hr and cooled to ambienttemperature. The reaction was quenched by addition of H₂O followed by15% NaOH(aq.) and then EtOAc. The organic layer was separated and thenfiltered through a short silica gel plug before being concentrated toyield 100 mg (99%) of the desired product (5) as a yellow oil. SeeCompound 6, FIG. 12F.

¹HNMR (300 MHz, CDCl₃) δ 7.40-7.24 (m, 6H), 7.06 (d, J=2.4, 1H), 6.70(d, J=15.9, 1H), 6.38 (dd, J=15.9, 5.4, 1H), 6.26 (d, J=9.9, 1H), 5.66(d, J=9.9, 1H), 5.50 (m, 1H), 1.44 (s, 3H), 1.42 (s, 3H); ¹³CNMR (75MHz, CDCb) δ 149.0, 136.9, 132.5, 132.0, 130.6, 129.5, 128.8, 128.8,128.3, 127.9, 126.8, 123.5, 121.6, 113.3, 77.4, 70.7, 28.3, 28.2; IR(NaCl, film) 3416 br cm⁻¹; HRMS (+TOF) 353.0536 calcd for C₁₁H₁₅O₃[M−H₂O]⁺. found: 353.0548. R_(f)=0.26 (9:1 hex./EtOAc).

Synthesis of(E)-1-(6-bromo-2,2-dimethyl-2H-chromen-8-yl)-3-phenylprop-2-en-1-one (6)

To a 50 ml round bottomed flask containing 1.41 g (3.80 mmol, 1 equiv.)(E)-1-(6-bromo-2,2-dimethyl-2H-chromen-8-yl)-3-phenylprop-2-en-1-oldissolved in 20 mL dry CH₂Ch was added 1.98 g (22.8 mmol, 6 equiv.) MnO₂and the reaction was stirred at ambient temperature for 2 hr. Thereaction was filtered through celite and concentrated. The resultingresidue was purified by silica gel flash chromatography eluting with19:1 to 4:1 25 hex./EtOAc yielding 1.25 g (89%) of the desired product(6) as a yellow oil. See Compound 8, FIG. 12F.

¹HNMR (300 MHz, CDCl₃) δ 7.70-7.39 (m, 8H), 7.22 (d, J=2.4, 1H), 6.31(d, J=9.9, 1H), 5.72 (d, J=9.9, 1H), 1.49 (s, 6H);

¹³CNMR (75 MHz, CDCl3) δ 190.6, 151.3, 143.4, 135.3, 132.3, 130.6,129.8, 129.2, 128.7, 128.5, 126.7, 124.2, 121.4, 113.2, 78.0, 28.5; IR(NaCl, film) 1654, 1603, 1435 cm⁻¹; HRMS (+TOP) 369.0485 calcd forC₂₀H₁₈BrO₂ [M+H]⁺. found: 369.0489. R_(f)=0.50 (4:1 hex./EtOAc).

Synthesis of Compound 8 (KAM-1)

To a 10 mL round bottomed flask containing 92 mg (0.25 mmol, 1 equiv.)(E)-1-(6-bromo-2,2-dimethyl-2H-chromen-8-yl)-3-phenylprop-2-en-1-one and83 mg (0.28 mmol, 1.1 equiv.) of Molander salt XX was added 5 9 mg(0.012 mmol, 0.05 equiv.) PdCb(dppf)-CH₂Ch and 245 mg (0.75 mmol, 3equiv.) Cs₂CO₃ followed by 1.5 mL dry PhMe and 0.5 mL H₂0. The reactionwas heated to 80° C. for 12 hr, filtered through cotton andconcentrated. Purification by silica gel flash chromatography elutingwith 9:1 hex./EtOAc yielded 78 mg (65%) of the desired product (8) as ayellow oil.

¹HNMR (300 MHz, CDCl₃) δ 8.10 (d, J=7.5, 2H), 7.71-7.20 (m, 14H), 6.76(d, J=2.4, 1H), 6.21 (d, J=9.9, 1H), 5.65 (d, J=9.9, 1H), 4.51 (t,J=7.8, 2H), 3.06 (t, J=7.8, 2H), 1.48 (s, 6H).

¹³CNMR (75 MHz, CDCl3): δ 191.9, 151.3, 142.6, 140.3, 135.6, 131.3,131.1, 130.8, 130.4, 130.1, 129.2, 128.5, 128.3, 127.2, 125.8, 123.1,122.5, 122.2, 120.6, 119.1, 108.8, 44.9, 34.3, 28.4. IR (NaCl, film)1655, 1596, 1485, 1453 cm⁻¹. HRMS (+TOF) 484.2271 calcd for C₃₄H₃₀NO₂[M+H]+. found: 484.2269. R_(f)=0.21 (9:1 hex./EtOAc).

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications, U.S. and foreign patents and patentapplications, are specifically and entirely incorporated by reference.The term comprising, where ever used, is intended to include the termsconsisting and consisting essentially of. Furthermore, the termscomprising, including, and containing are not intended to be limiting.It is intended that the specification and examples be consideredexemplary only with the true scope and spirit of the invention indicatedby the following claims.

1. A method of treating or preventing a disease or disorder associatedwith aberrant PKC delta activity comprising: formulating a pharmaceuticcomposition comprising a compound of the formula:

wherein: R₁ is selected from the group consisting of H and a loweralkyl; R₂ and R₃ are each independently selected from the groupconsisting of H, OH and OR; A, B, C, D, W, X, Y, and Z are eachindependently selected from the group consisting of N and CH; G isselected from the group consisting of O, NR, S, and CH₂, R is selectedfrom the group consisting of H, a lower alkyl and aryl; and n is aninteger selected from the group consisting of 1, 2, 3, and 4;administering a therapeutically effective amount of the pharmaceuticalcomposition to a patient with the disease or disorder associated withaberrant PKC delta activity.
 2. The method of claim 1, wherein thedisease or disorder is a cell proliferative disease, a cancer, a tumor,a benign neoplasm, an allergic disease, an inflammatory disease, or anautoimmune disease.
 3. The method of claim 1, wherein the pharmaceuticalcomposition inhibits PKC delta activity of cells of the patient.
 4. Themethod of claim 1, wherein cells associated with the disease or disorderhave aberrantly increased Ras signaling that is inhibited byadministration of the pharmaceutical composition.
 5. The method of claim1, wherein the disease or disorder is selected from the group consistingof diseases of the central nervous system (CNS), neurodegenerativediseases, vascular diseases, restenosis, musculoskeletal diseases,cardiovascular diseases, stroke, pulmonary diseases, gastric diseases,infectious diseases, fibrotic diseases, chronic kidney disease, liverfibrosis, pulmonary/lung fibrosis, systemic sclerosis, idiopathicpulmonary fibrosis, cystic fibrosis, cirrhosis, endomyocardial fibrosis;mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis,progressive massive fibrosis, coal workers' pneumoconiosis, nephrogenicsystemic fibrosis, Crohn's disease, Keloid, myocardial infarction,scleroderma/systemic sclerosis, post-radiation fibrosis, drug-inducedfibrosis and combinations thereof.
 6. The method of claim 1, wherein thepharmaceutical composition comprises one or more excipients, solvents,diluents, liquid vehicles, dispersions, suspension aids, surface activeagents, isotonic agents, thickening agents, emulsifying agents,preservatives, antioxidants, solid binders, lubricants, pharmaceuticallyacceptable salts and/or combinations thereof.
 7. The method of claim 6,wherein the pharmaceutically acceptable salts are selected from thegroup consisting of ammonium, calcium, sodium, and combinations thereof.8. The method of claim 1, wherein R₁, R₂ and R₃ are each independently Hor OR, and wherein R is H or CH₃.
 9. The method of claim 1, wherein R₁,R₂ and R₃ are each H.
 10. The method of claim 1, wherein A, B, C, D, W,X, Y, and Z are each CH, and G is CH₂.
 11. The method of claim 1,wherein n is 1 or
 2. 12. The method of claim 1, wherein thepharmaceutical composition is administered orally, parentally ortopically in one or more doses.
 13. The method of claim 1, where thepharmaceutical composition is administered by intravenous injection. 14.The method of claim 1, wherein the therapeutically effective amount ofthe pharmaceutical composition is that amount effective for killing orinhibiting aberrant cell proliferation.
 15. The method of claim 1,wherein the therapeutically effective amount is from 2 to 500 mg/kg ofbody weight administered one or more times per day.
 16. The method ofclaim 1, wherein the patient is a mammal or a human.
 17. A method ofmanufacturing a pharmaceutical composition comprising: synthesizing acompound of the formula:

wherein: R₁ is selected from the group consisting of H and a loweralkyl; R₂ and R₃ are each independently selected from the groupconsisting of H, OH and OR; A, B, C, D, W, X, Y, and Z are eachindependently selected from the group consisting of N and CH; G isselected from the group consisting of O, NR, S, and CH₂, R is selectedfrom the group consisting of H, a lower alkyl and aryl; and n is aninteger selected from the group consisting of 1, 2, 3, and 4; andformulating the compound as a pharmaceutical composition.
 18. The methodof claim 17, wherein R₁, R₂ and R₃ are each independently H or OR, andwherein R is H or CH₃.
 19. The method of claim 17, wherein R₁, R₂ and R₃are each H.
 20. The method of claim 17, wherein A, B, C, D, W, X, Y, andZ are each CH, and G is CH₂.
 21. The method of claim 17, wherein n is 1or
 2. 22. The method of claim 17, wherein formulating comprises addingone or more excipients, solvents, diluents, liquid vehicles,dispersions, suspension aids, surface active agents, isotonic agents,thickening agents, emulsifying agents, preservatives, antioxidants,solid binders, lubricants, pharmaceutically acceptable salts and/orcombinations thereof.
 23. The method of claim 22, wherein thepharmaceutically acceptable salts are selected from the group consistingof ammonium, calcium, sodium, and/or combinations thereof.
 24. Themethod of claim 17, further comprises apportioning the pharmaceuticalcomposition as one or more therapeutically effective amounts foradministration to a patient orally, parentally or topically.
 25. Themethod of claim 24, wherein the one or more therapeutically effectiveamounts comprises from 2 to 500 mg of compound per kg of body weight ofthe patient.
 26. The method of claim 25, wherein the patient is a mammalor a human.
 27. A method comprising: assessing cells of a patient foraberrant PKC delta activity; and determining a therapeutically effectiveamount for inhibiting PKC delta activity of a pharmaceutical compositioncontaining a compound of the formula:

wherein: R₁ is selected from the group consisting of H and a loweralkyl; R₂ and R₃ are each independently selected from the groupconsisting of H, OH and OR; A, B, C, D, W, X, Y, and Z are eachindependently selected from the group consisting of N and CH; G isselected from the group consisting of O, NR, S, and CH₂, R is selectedfrom the group consisting of H, a lower alkyl and aryl; and n is aninteger selected from the group consisting of 1, 2, 3, and
 4. 28. Themethod of claim 27, wherein R₁, R₂ and R₃ are each independently H orOR, and wherein R is H or CH₃.
 29. The method of claim 27, wherein R₁,R₂ and R₃ are each H.
 30. The method of claim 27, wherein A, B, C, D, W,X, Y, and Z are each CH, and G is CH₂.
 31. The method of claim 27,wherein n is 1 or
 2. 32. The method of claim 27, wherein the cells areaberrantly proliferating cells.
 33. The method of claim 32, wherein theaberrantly proliferating cells are cancer cells.
 34. The method of claim32, where the therapeutically effective amount is that amount whichinhibits PKC delta activity, inhibits proliferation and/or kills theaberrantly proliferating cells.
 35. The method of claim 27, furthercomprising administering the therapeutically effective amount of thepharmaceutical composition to the cells of the patient orally,parentally or topically and in one or more doses.
 36. The method ofclaim 35, where the therapeutically effective amount is from 2 to 500mg/kg of body weight of the patient and the pharmaceutical compositionis administered intravenously in one or more doses per day.